Glutamate transporter associated proteins and methods of use thereof

ABSTRACT

Glutamate Transporter Associated Proteins and nucleotide encoding Glutamate Transporter Associated Proteins are provided. Also provided is a method for identifying a compound that modulates a cellular response mediated by a Glutamate Transporter Associated Protein. A method is further provided for identifying a compound that inhibits an interaction between a Glutamate Transporter Associated Protein and a glutamate transporter protein. A method is provided for treating a disorder associated with glutamate transport.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e)(1) to U.S.Provisional Application Ser. No. 60/161,007, filed Oct. 23, 1999, and toU.S. Provisional Application Ser. No. 60/206,157, filed on May 22, 2000,each herein incorporated by reference in their entirety.

GRANT INFORMATION

This invention was made with Government support under NS33958 andNS70151, awarded, by the National Institutes of Health (NINDS). TheGovernment may have certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to protein-protein interactionsand more specifically to Glutamate Transporter Associated Proteinsinvolved in mediating glutamate transport, chloride transport andcytoskeletal stability and their association with glutamate transporterproteins.

2. Background Information

Glutamate is the major excitatory neurotransmitter in the mammaliancentral nervous system, acting on postsynaptic ionotropic glutamatereceptors (particularly NMDA and AMPA receptors). In addition, glutamatestimulates a subset of metabotropic glutamate receptors (particularlythe group I metabotropic glutamate receptors mGluR1a and mGluR5)concentrated in the postsynaptic membrane. The timely removal ofglutamate from the synaptic cleft is critical to preventingdesensitization resulting from continued exposure of the postsynapticreceptors to glutamate. Removal of glutamate from the synaptic cleft ismediated by a class of molecules known as glutamate transporter proteinslocated on surrounding astroglia and neurons. Five distinct,high-affinity, sodium-dependent glutamate transporters have beenidentified in animal and human central nervous system. Rat GLAST, GLT-1,EAAC1 (EAAT1, EAAT2 and EATT3, respectively, in human), EAAT4 and EAAT5differ in structure, pharmacological properties and tissue distribution.

Glutamate transport is a sodium- and potassium-coupled process capableof concentrating intracellular glutamate up to 10,000-fold compared withthe extracellular environment. The stoichiometry of the process has beenstudied and at several models exist proposing various ionic exchanges.In one model derived from salamander retinal glial cells, the transportprocess is coupled to the co-transport of two sodium ions and thecounter-transport of one potassium ion and one hydroxyl ion. (Bouvier etal. (1992), Nature 360:471-474). Another model proposes that with EAAC1,one glutamate is co-transported with three sodium ions and one hydrogenion, with the counter-transport of one potassium ion (Zerangue et al,Nautre (1996) 383:634-637). Yet another study suggests that two sodiumions are co-transported with one glutamate molecule (Hart et al.,Science (1998) 280:2112-2114).

The cloning of glutamate transporter subtypes and detailedelectrophysiological studies of these proteins reveals that glutamatetransporters also possess channel-like properties. The conduct chlorideflux is not thermodynamically coupled to substrate transport, althoughat transportable substrate is required for the chloride conductance. Thebinding of glutamate to the transporter may change its conformationalstate to form the chloride channel.

In addition to their possible role in development and learning (due totheir potential for modulating normal synaptic transmission), theregulation of synaptic glutamate transporters is likely to play animportant role in acute and chronic neurological processes. They can beinvolved through the disruption of synaptic transmission as well asthrough glutamate mediated excitotoxicity. Several diseases areassociated with disruptions in glutamate transport.

Loss of cerebellar Purkinje cell is the hall mark of several inheritedneurodegenerative diseases, including the trinucleotide repeat diseasessuch as spinocerebellar ataxia type 1 (SCA1), and is commonly associatedwith neurotoxicity of chronic ethanol ingestion, and with certainparaneoplastic neurological disorders. Although the molecular event thatinitiates the disease is known—a trinucleotide repeat—the cellularmechanisms responsible for Purkinje cell degeneration is not known. Theselective loss of glutamate transporters such as EAAT4 could make theprotein an attractive candidate for a downstream event.

Similarly, dysregulation of glutamate transporter EAAC1 could also havepathological consequences. EAAC1 has the unusual localization to GABApre-synaptic terminals. This transport could serve as a precursortransporter, supplying extracellular glutamate for GABA re-synthesis.GABA normally is synthesized, via glutamate amino decarboxylase, fromglutamate. The source of this glutamate has been traditionally thoughtto be cellular glutamate. However, the unique localization of theglutamate transporter to GABA terminals suggests that these transporterssupply precurser glutamate for GABA re-synthesis. Thus, EAAC1 couldserve as an important step in GABAergic neurotransmission. Modulation ofGABAergic metabolism is associated with a number of neurologicaldisorders, including epilepsy, tremors, and spasticity. In addition,some theories of schizophrenia include disturbances of glutamate andGABA metabolism.

Accordingly, there is a need in the art for compounds that regulateglutamate transport and in particular, compounds and molecules thatinteract with glutamate transporter proteins.

SUMMARY OF THE INVENTION

The present invention provides a family of proteins that interact withglutamate transporter proteins. Through their interaction with glutamatetransporter proteins, Glutamate Transporter Associated Proteins modulateglutamate transport, and also effect cytoskeletal organization andstability as well as chloride flux.

In one embodiment of the invention, there is provided a substantiallypure polypeptide characterized as modulating intracellular glutamatetransport, interacting with a glutamate transporter protein, and havingan expression pattern in the brain. In addition, the polypeptide canhave at least one PDZ domain, at least one regulatory G-protein domain,at lest one pleckstrin homology domain, at least one proline-rich domainand at least one guanine exchange factor domain. The polypeptide canhave at least one pleckstrin homology domain, at least one spectrinrepeat and at least one α-actinin domain.

In an additional embodiment of the invention, there is provided asubstantially pure polypeptide characterized as modulating intracellularglutamate transport; interacting with a glutamate transporter protein;having an expression pattern in neural non-neuronal tissues; having atleast one kinase C domains; having four transmembrane domains; and beinghydrophobic.

In another embodiment of the invention, there is provided asubstantially pure polypeptide having an amino acid sequence as setforth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or conservative variantsthereof.

In still another embodiment of the invention, there is provided anisolated polynucleotide selected from the group consisting of: (a) apolynucleotide encoding a polypeptide having an amino acid sequence asset forth in SEQ ID NO:2; (b) a polynucleotide of (a), wherein T can beU; (c) a polynucleotide complementary to (a) or (b); (d) apolynucleotide having a nucleotide sequence as set forth in SEQ ID NO:1; (e) degenerate variants of (a), (b), (c) or (d); and (f) a fragmentof (a), (b), (c), (d) or (e) having at least 15 base pairs andhybridizes to a polynucleotide encoding a polypeptide as set forth inSEQ ID NO:2.

In yet another embodiment of the invention, there is provided anisolated polynucleotide selected from the group consisting of: (a) apolynucleotide encoding a polypeptide having an amino acid sequence asset forth in SEQ ID NO:4; (b) a polynucleotide of (a), wherein T can beU; (c) a polynucleotide complementary to (a) or (b); (d) apolynucleotide having a nucleotide sequence as set forth in SEQ ID NO:3;(e) degenerate variants of (a), (b), (c) or (d); and (e) a fragment of(a), (b), (c), (d) or (e) having at least 15 base pairs and hybridizesto a polynucleotide encoding a polypeptide as set forth in SEQ ID NO:4.

In still another embodiment of the invention, there is provided anisolated polynucleotide selected from the group consisting of: (a) apolynucleotide encoding a polypeptide having an amino acid sequence asset forth in SEQ ID NO:6; (b) a polynucleotide of (a), wherein T can beU; (c) a polynucleotide complementary to (a) or (b); (d) apolynucleotide having a nucleotide sequence as set forth in SEQ ID NO:5;(e) degenerate variants of (a), (b), (c) or (d); and (f) a fragment of(a), (b), (c), (d) or (e) having at least 15 base pairs and hybridizesto a polynucleotide encoding a polypeptide as set forth in SEQ ID NO:6.

In still a further embodiment of the invention, there is provided anantibody that binds to a Glutamate Transporter Associated Protein orbinds to immunoreactive fragments thereof. The antibody can bepolyclonal or monoclonal.

In yet another embodiment of the invention, there is provided anexpression vector comprising a polynucleotide encoding GlutamateTransporter Associated Protein, e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, or complementary nucleotides thereof and fragments thereof. Thevectors can be virus derived or plasmid derived.

In another embodiment of the invention, there is provided a method forproducing a Glutamate Transporter Associated Protein polypeptide byculturing a host cell containing a nucleotide encoding a GlutamateTransporter Associated Protein under conditions suitable for theexpression of the polypeptide and recovering the polypeptide from thehost cell culture.

In another embodiment of the invention, there is provided asubstantially pure polypeptide that interacts with the amino acidsequence QEAELTLP (SEQ ID NO:9) or amino acid sequence GRGGNESVM (SEQ IDNO:10).

In still another embodiment of the invention, there is provided asubstantially pure polypeptide that interacts with the amino acidsequence set forth in SEQ ID NO: 12.

In still another embodiment of the invention, there is provided asubstantially pure polypeptide that interacts with the amino acidsequence set forth in SEQ ID NO: 13.

In an addition embodiment of the invention, there is provided a methodfor identifying a compound that modulates a cellular response mediatedby a Glutamate Transporter Associated Protein. The method includesincubating the compound with a cell expressing a Glutamate TransporterAssociated Protein and a glutamate transporter protein under conditionssufficient to permit the components to interact and comparing a cellularresponse in the cell incubated with the compound with the cellularresponse of a cell not incubated with the compound.

In yet another embodiment of the invention, there is provided a methodfor identifying a compound that inhibits an interaction between aGlutamate Transporter Associated Protein and a glutamate transporterprotein. The method includes contacting a Glutamate TransporterAssociated Protein with a glutamate transporter protein in the presenceof the compound and comparing the formation of a Glutamate TransporterAssociated Protein-glutamate transporter protein complex in the presenceof the compound with a formation of the complex in the absence of thecompound.

In still another embodiment of the invention, there is provided atransgenic non-human animal having a transgene that expresses aGlutamate Transporter Associated Protein chromosomally integrated intothe germ cells of the animal. An embodiment of the invention provides amethod for producing such transgenic animals.

In another embodiment of the invention, there is provided a computerreadable medium having stored thereon a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5 andsequences substantially identical thereto, or a polypeptide sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6 and sequences substantially identical thereto.

In another embodiment of the invention, there is provided a computersystem comprising a processor and a data storage device wherein saiddata storage device has stored thereon a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 andsequences substantially identical thereto, or a polypeptide sequenceselected from the group consisting SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6and sequences substantially identical thereto.

In yet another embodiment of the invention, there is provided a methodfor comparing a first sequence to a reference sequence wherein saidfirst sequence is a nucleic acid sequence selected from the groupconsisting SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5 and sequencessubstantially identical thereto, or a polypeptide sequence selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 andsequences substantially identical thereto. The method comprises readingthe first sequence and the reference sequence through use of a computerprogram which compares sequences, and determining differences betweenthe first sequence and the reference sequence with the computer program.

In yet another embodiment of the invention there is provided a methodfor identifying a feature in a sequence wherein the sequence is selectedfrom the group consisting of a nucleic acid sequence SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5 sequences substantially identical thereto, or apolypeptide sequence SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and sequencessubstantially identical thereto. The method includes reading thesequence through the use of a computer program which identifies featuresin sequences and identifying features in the sequences with the computerprogram.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show schematic representations of GTRAP4-41 andGTRAP4-48, respectively.

FIG. 2 shows overlapping deletion mutants of the carboxy terminus ofEAAT4 used to identify domains interacting with GTRAP4-41 and GTRAP4-48.

FIG. 3A shows the effect of GTRAP4-41 and GTRAP4-48 on sodium-dependentglutamate uptake in transfected HEK-rEAAT4 cells. FIG. 3B shows kineticdata which demonstrates that GTRAP4-41, in the presence of EAAT4,increases the V_(max) of glutamate uptake.

FIG. 4A shows the effect of GTRAP3-18 on sodium-dependent glutamatetransport in transfected HEK-293 cells. FIG. 4B shows that the effect ofGTRAP 3-18 on EAAC 1-mediated glutamate transport is specific.

FIGS. 5A and 5B show the effect of Glutamate Transporter AssociatedProteins (GTRAPs) on glutamate transporter protein expression.

FIGS. 6A-C show the effect of GTRAPs on glutamate transporter proteinactivity.

FIGS. 7A and 7B show the interaction between GTRAP4-48 and RhoGEF.

FIGS. 8A-C show the effects of GTRAP3-18 antisense oligonucleotide onglutamate transport.

FIGS. 9A-E show the effect of retinoic acid on GTRAP3-18-mediatedglutamate transport.

FIG. 10 is a flow diagram illustrating a computer system, dataretrieving device and display.

FIG. 11 is a flow diagram illustrating one embodiment of process 200 forcomparing a new nucleotide or protein sequence with a database ofsequences in order to determine the homology levels between the newsequence and the sequences in the database.

FIG. 12 is a flow diagram illustrating one embodiment of a process 250in a computer for determining whether two sequences are homologous.

FIG. 13 is a flow diagram illustrating one embodiment of a process 300for comparing features in polynucleotide and polypeptide sequences.

FIG. 14 (A-C) shows a nucleic acid sequence of a polynucleotide encodingGTRAP4-41.

FIG. 15 shows an amino acid sequence of GTRAP4-41.

FIG. 16 (A and B) shows a nucleic acid sequence of a polynucleotideencoding GTRAP4-48.

FIG. 17 shows an amino acid sequence of GTRAP4-48.

FIG. 18 shows a nucleic acid sequence of a polynucleotide encodingGTRAP3-18.

FIG. 19 shows an amino acid sequence of GTRAP3-18.

FIG. 20 (A and B) shows a nucleic acid sequence of a polynucleotideencoding PCTAIRE-1.

FIG. 21 shows an amino acid sequence of PCTAIRE-1a.

FIG. 22 shows an amino acid sequence of PCTAIRE1b.

DETAILED DESCRIPTION OF THE INVENTION

The identification of molecules regulating the transport ofneurotransmitters is central to understanding the mechanisms of neuralactivity, synaptic plasticity and learning. Efficient and rapid removalof neurotransmitters from the synaptic cleft by neurotransmittertransporters is critical to synaptic transmission. Re-uptake ofglutamate by glutamate transporters both terminates the synaptic actionof glutamate, thereby preventing glutamate-mediated exotoxicity andrecaptures glutamate molecules for possible reuse.

Accordingly, one embodiment of the invention provides a substantiallypure polypeptide characterized as modulating intracellular glutamatetransport, interacting with a glutamate transporter protein and havingan expression pattern in the brain. A polypeptide molecule having suchcharacteristics is known as a Glutamate Transporter Associated Protein(GTRAP). Glutamate Transporter Associated Proteins can be furthercharacterized as having at least one PDZ domain, having at least oneregulatory G-protein domain, having at least one pleckstrin homologydomain, having at least one proline-rich domain, and having at least oneguanine exchange factor domain. Glutamate Transporter Associated Proteincan also be characterized as having at least one pleckstrin homologydomain, having at least one spectrin repeat, and having at least onea-actinin domain.

Glutamate Transporter Associated Proteins modulate glutamate transport.Glutamate transport refers to the active movement of glutamate across acellular membrane. Glutamate transport is an essential component ofcentral nervous system glutamatergic neurotransmission. For example,glutamate transport is essential in the inactivation of synapticallyreleased glutamate and the prevention of excitotoxicity. Theconcentration of glutamate is higher in the terminal than in thesynaptic cleft, even following neurotransmitter release. Nonetheless,the transporters take up glutamate from the synaptic cleft and transportit into the cell. Glutamate transporters also serve to bring glutamateinto the cell for use in cellular metabolism, e.g. provide glutamate fornew synthesis of neurotransmitter GABA. GTRAPs associated with sometypes of glutamate transporter protein, for example, glutamatetransporter protein EAAT4, stimulate glutamate transport. GTRAPsassociated with other types of glutamate transporter proteins, forexample, EAAC1, inhibit glutamate transport. While not wishing to bebound to any one mechanism, the modulation in transport appears to beeffected through a change in Vmax or a change in Km (see Examplessection). Glutamate transporter proteins can signal messages to the cellabout transport activities e.g. GTRAP48 activate G-protein signaling].

Glutamate Transporter Associated Proteins share several common features.All GTRAPs are able to interact with at least one glutamate transporterprotein. Glutamate transporter proteins include GLAST, GLT-1, EAAC1,EAAT1, EAAT2, EAAT3, EAAT4 and EAAT5. Glutamate transporters share over50% amino acid sequence identity with each other, and display almostidentical hydrophobic profiles including six prominent hydrophobicpeaks, followed by a small hydrophobic peak and long hydrophobicstretch. The proteins are generally 500 to 600 amino acids in length,with high conservation of sequence in the transmembrane domain. Thecarboxyl and amino terminal domains are intracellular and have the leastsequence conservation among all transporters. Less is known about thegenomic structure of the transporter proteins. The glutamate transporterfamily is quite distinct in structure from the 12 transmembrane α-helixarrangement of another sodium- and chloride-dependent transporter familyrelated to dopamine and serotonin transport. The glutamate transporterfamily transports L-glutamate, D-aspartate and L-aspartate and someother acidic amino acids such as threo-β-hydroxyaspartate (THA) andcysteate. However, the transporters display distinct properties insubstrate or inhibitor selectivity, e.g. dihydrokainate is a specificinhibitor of GLT-1 and EAAC1 transports cysteine with much higheraffinity than the other transporters. Various studies have suggestedthat transporters may form homomultimers, perhaps dimers, butphysiological transport may only require monomers of the protein.

Immunohistochemical studies show that GLAST and GLT-1 (EAAT1 and EAAT2)are localized primarily in astrocytes. In the adult CNS, GLT-1 is widelydistributed throughout the brain and spinal cord in astroglial cellbodies and processes, while GLAST protein is localized in glial cells ofcerebellar molecular and granule cell layers, and in some astrogliathroughout the brain. Double labeling post-embedding electronmicroscopic immunocytochemistry shows the two glial transporters, GLT-1and GLAST, expressed in the same cell membrane. Each protein formsoligomeric complexes but GLT-1 and GLAST may not complex with eachother. Antisense knock-down studies show that these two glialtransporters are responsible for over 80% of glutamate uptake in thebrain, an observation later confirmed in GLT-1 null mice. Quantitativeimmunoblotting and electron microscopy indicate that the glialtransporters are quite abundant; GLAST and GLT-1 respectively, are 2300and 8500 molecules per μm² in CA1 hippocampus membrane, and 4700 and 740molecules per μm² in the cerebellar molecular layer.

Developmental studies reveal differential expression of GLT-1 and GLASTmRNA and protein. Initially expression of GLAST predominates throughoutthe CNS, followed by a shift in expression to the cerebellum, whereasGLT-1 expression remains throughout most of the CNS. A dramaticup-regulation of GLT-1 gene expression at post-natal day 14 coincideswith the post-natal development of glutamatergic transmission in thecortex.

GLT-1 mRNA and protein can, under certain conditions be found inneurons, e.g. cultured hippocampal neurons. Transiently localized GLT-1on growing axons and axon pathways can also be detected. Additionalstudies in models of ischemic brain injury and in fetal ovine brainsuggest rare neuronal expression of GLT-1 as well.

EAAC1 and EAAT4 are neuronal transporters. EAAC1 immunoreactivity isparticularly high in regions such as the hippocampus, cerebellum andbasal ganglia. It is widely distributed in neurons such as largecortical pyramidal neurons, and is also present in non-glutamatergicneurons including GABAergic cerebellar Purkinje cells. Ultra-structuralstudies suggest that EAAC1 is not a presynaptic transporter ofglutamatergic neurons. In fact, EAAC1 appears to be primarily localizedin the somatodendritic compartment, and is already expressed at stagespreceding synaptic contact formation. Rarely, EAAC1 is found inpre-synaptic terminals, which are always inhibitory (e.g. GABAergic).Ultra structurally, EAAC1 is present in dendrites and somas. DetailedEM-gold studies of synapses indicate that the protein is most oftenperi-synaptic in location, like EAAT4. EAAC1 is also widely expressedoutside the central nervous system, so it may serve metabolic functionsin neurons. For example, it may provide glutamate for resynthesis ofGABA in GABAergic terminals, where the protein has been localized(Rothstein, et al. (1994) Neuron 13:713-725, herein incorporated byreference in its entirety). In fact, studies using antisenseoligonucleotides to inhibit EAAC1 suggest that this transporter may, inpart, regulate GABA synthesis.

EAAT4 is largely expressed in the cerebellum with very faint levels ofexpression in hippocampus, neocortex, striatum, brain stem and thalamus,in both the adult human and rat CNS. EAAT4 is present at lowconcentrations in the synaptic membrane, but is highly enriched in theparts of the dendritic and spine membranes facing astrocytes. Afunctional relationship may exist between EAAT4 and the glialtransporters, and that EAAT4, having a prominent C1⁻-channel property,may function as a combined transporter and inhibitory glutamatereceptor. The average density of EAAT4 protein in the Purkinje cellmembrane has been calculated to be 1800 molecules per η².Immunohistochemical as well as immunoblot analysis demonstrates thatduring development EAAT4 protein is expressed in the human cerebellumboth pre- and post-natally, while its expression in the frontal cortexis restricted to fetal stages. In the cerebellum, Purkinje cells showfaint EAAT4 immunoreactivity at gestation week 17. However, EAAT4expression becomes increasingly intense from gestation week 23 to theinfantile period. After the late infantile period, EAAT4immunoreactivity shows the same pattern as in adults. The intracellularlocalization of EAAT4 also changes with development. In the earlyembryonic period, EAAT immunoreactivity is found in the short processesof the Purkinje cells, while in the late fetal to early infantileperiods, EAAT4 immunoreactivity is found in the cell bodies anddendrites, and in the late infantile period, it is found in the spines.

Glutamate transporters and glutamate receptors are compartmentalized inand around the synaptic cleft and proteins capable of glutamate receptormembrane targeting and the epitopes responsible for these events areknown. For example, three cytoplasmic molecules have been recentlyidentified which bind to the final eight amino acids in the C-terminusof GluR2 and GluR3, but not to GluR1 or NR1. These molecules, named GRIPand ABP are all synaptically localized in the hippocampus and containone or more PDZ domains, protein binding motifs of between 70 and 90amino acids which have recently been implicated in the localization ofother highly regulated proteins. None of these molecules interacts orregulates glutamate transporters.

Several studies document a role for neurons in modulating the expressionand activity of glutamate transporters. Pathway lesion studies suggestthat neurons can influence the astroglial (but not neuronal) expressionof glutamate transporter subtypes. This has been validated in vitro,where astroglial (EAAT2) expression in cultured astrocytes appears todepend on neurons, most likely secreted factors, including glutamateitself. In fact, a number of trophic factors that modulate EAAT2expression in various in vitro preparations have been identified.Protein kinase C phosphorylation of EAAT2 (GLT-1) has also been found tostimulate transport. Transporters can also be directly regulated throughother signaling pathways. Activity of EAAC1 (and GABA and serotonintransporters) can be regulated through expression at the cell surface,via regulated cellular trafficking, occurring in part through proteinkinase C and phosphatidylinositol 3-kinase pathways.

Glutamate Transporter Associated Proteins have an expression pattern inbrain tissue. Immunofluorescence staining of brain tissue reveals apattern of GTRAP immunoreactivity in brain tissue. Prominentimmunolocalization is observed in the cerebellar cortex, especially inPurkinje cell somas and dendrites with no axonal localization.Expression is also observed in other brain regions including striatum,hippocampus and thalamus.

Expression of certain Glutamate Transporter Associated Proteins isobserved outside the brain. For example, GTRAP3-18 is expressed in theliver, kidney, heart, muscle as well as in the central nervous system.

Glutamate Transporter Associated Proteins can include at least one PDZdomain, at least one regulatory G-protein domain, at least onepleckstrin homology domain (PH), at least one proline-rich domain, atleast one guanine exchange factor domain (Dbl), at least one spectrinrepeat and at least one a-actinin domain. Methods to identify suchdomains are known to those of skill in the art. For example, computerprograms that compare invention nucleic acid and amino acid sequences tonucleic acid and amino acid sequences, and identify regions of homologycan be used to identify such domains.

Exemplary Glutamate Transporter Associated Proteins of the inventioninclude sequences as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6and conservative variants thereof. The terms “conservative variation”and “substantially similar” as used herein denotes the replacement of anamino acid residue by another, biologically similar residue. Examples ofconservative variations include the substitution of one hydrophobicresidue such as isoleucine, valine, leucine or methionine for another,or the substitution of one polar residue for another, such as thesubstitution of arginine for lysine, glutamic acid for aspartic acid, orglutamine for asparagine, and the like. The terms “conservativevariation” and “substantially similar” also include the use of asubstituted amino acid in place of an unsubstituted parent amino acidprovided that antibodies raised to the substituted polypeptide alsoimmunoreact with the unsubstituted polypeptide.

Also contemplated by the invention are polypeptides that share at least90% sequence homology to the polypeptide sequences set forth as SEQ IDNO:2, SEQ ID NO:4 and SEQ ID NO:6. Sequence homology can be determinedby those of skill in the art, for example, by computer programs thatcompare sequences such as Blast.

Exemplary polynucleotides encoding a Glutamate Transporter AssociatedProteins are set forth as SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5 and SEQID NO:7. The term “polynucleotide”, “nucleic acid”, “nucleic acidsequence”, or “nucleic acid molecule” refers to a polymeric form ofnucleotides at least 10 bases in length. By “isolated polynucleotide” ismeant a polynucleotide that is not immediately contiguous with both ofthe coding sequences with which it is immediately contiguous (one on the5′ end and one on the 3′ end) in the naturally occurring genome of theorganism from which it is derived. The term therefore includes, forexample, a recombinant DNA which is incorporated into a vector; into anautonomously replicating plasmid or virus; or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g., acDNA) independent of other sequences. The nucleotides of the inventioncan be deoxyribonucleotides, ribonucleotides in which uracil (U) ispresent in place of thymine (T), or modified forms of either nucleotide.The nucleotides of the invention can be complementary to thedeoxynucleotides or to the ribonucleotides. A polynucleotide encoding aGlutamate Transporter Associated Protein includes “degenerate variants”,sequences that are degenerate as a result of the genetic code. There are20 natural amino acids, most of which are specified by more than onecodon. Therefore, all degenerate nucleotide sequences are included inthe invention as long as the amino acid sequence of a polypeptideencoded by the nucleotide sequence of SEQ ID NO: 1,SEQ ID NO:3, SEQ IDNO:5 or SEQ ID NO:7 is functionally unchanged.

A nucleic acid molecule encoding a Glutamate Transporter AssociatedProtein includes sequences encoding functional Glutamate TransporterAssociated Protein polypeptides as well as functional fragments thereof.As used herein, the term “functional polypeptide” refers to apolypeptide which possesses biological function or activity which isidentified through a defined functional assay, and which is associatedwith a particular biologic, morphologic, or phenotypic alteration in thecell. The term “functional fragments of Glutamate Transporter AssociatedProtein,” refers to fragments of a Glutamate Transporter AssociatedProtein that retain a Glutamate Transporter Associated Protein activity,e.g., the ability to interact with a glutamate transporter protein,modulate intracellular glutamate transport, and the like. Additionally,functional Glutamate Transporter Associated Protein fragments may act ascompetitive inhibitors of Glutamate Transporter Associated Proteinbinding to a glutamate transporter protein, for example. Biologicallyfunctional fragments can vary in size from a polypeptide fragment assmall as an epitope capable of binding an antibody molecule to a largepolypeptide capable of participating in the characteristic induction orprogramming of phenotypic changes within a cell. Nucleotide fragments ofthe invention have at least 15 base pairs and hybridize to apolynucleotide encoding a polypeptide as set forth in SEQ ID NO:2, SEQID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:22.

Further embodiments of the invention provide isolated polynucleotides,wherein the nucleotide is at least 15 base pairs in length whichhybridizes under moderately to highly stringent conditions to DNAencoding a polypeptide as set forth in SEQ ID NO:2 or to DNA encoding apolypeptide as set forth in SEQ ID NO:4, or SEQ ID NO:6. In nucleic acidhybridization reactions, the conditions used to achieve a particularlevel of stringency will vary, depending on the nature of the nucleicacids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (e.g., GC v. ATcontent), and nucleic acid type (e.g., RNA v. DNA) of the hybridizingregions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.

An example of progressively higher stringency conditions is as follows:2×SSC/0.1% SDS at about room temperature (hybridization conditions);0.2×SSC/0.1% SDS at about room temperature (low stringency conditions);0.2×SSC/0.1% SDS at about 42° C. (moderately stringent conditions); and0.1×SSC at about 68° C. (highly stringent conditions). Washing can becarried out using only one of these conditions, e.g., high stringencyconditions, or each of the conditions can be used, e.g., for 10-15minutes each, in the order listed above, repeating any or all of thesteps listed. However, as mentioned above, optimal conditions will vary,depending on the particular hybridization reaction involved, and can bedetermined empirically.

Antibodies of the invention may bind to Glutamate Transporter AssociatedProteins provided by the invention to prevent normal interactions ofGlutamate Transporter Associated Proteins. Binding of antibodies toGlutamate Transporter Associated Protein can interfere with for example,glutamate transport, with cytoskeletal stability by interfering withintracellular protein binding, with expression patterns of GlutamateTransporter Associated Proteins or with interactions with glutamatetransporter proteins. Furthermore, binding of antibodies to GlutamateTransporter Associated Proteins can interfere with the localization ofglutamate transporter proteins on cellular membranes.

The antibodies of the invention can be used in any subject in which itis desirable to administer in vitro or in vivo immunodiagnosis orimmunotherapy. The antibodies of the invention are suited for use, forexample, in immunoassays in which they can be utilized in liquid phaseor bound to a solid phase carrier. In addition, the antibodies in theseimmunoassays can be detectably labeled in various ways. Examples oftypes of immunoassays which can utilize antibodies of the invention arecompetitive and non-competitive immunoassays in either a direct orindirect format. Examples of such immunoassays are the radioimmunoassay(RIA), the enzyme-linked immunosorbant assay (ELISA) and the sandwich(immunometric) assay. Detection of the antigens using the antibodies ofthe invention can be done utilizing immunoassays which are run in eitherthe forward, reverse, or simultaneous modes, includingimmunohistochemical assays on physiological samples. Those of skill inthe art will know, or can readily discern, other immunoassay formatswithout undue experimentation.

The term “antibody” as used in this invention includes intact moleculesas well as fragments thereof, such as Fab, F(ab′)2, and Fv which arecapable of binding to an epitopic determinant present in an inventionpolypeptide. Such antibody fragments retain some ability to selectivelybind with its antigen or receptor.

Methods of making these fragments are known in the art. (See forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1988), incorporated herein by reference).Monoclonal antibodies are made from antigen containing fragments of theprotein by methods well known to those skilled in the art (Kohler &Milstein, Nature 256:495 (1975); Coligan et al., sections 2.5.1-2.6.7;and Harlow et al., Antibodies: A Laboratory Manual, page 726 (ColdSpring Harbor Pub. 1988), which are hereby incorporated by reference.Briefly, monoclonal antibodies can be obtained by injecting mice with acomposition comprising an antigen/ligand, verifying the presence ofantibody production by analyzing a serum sample, removing the spleen toobtain B lymphocytes, fusing the B lymphocytes with myeloma cells toproduce hybridomas, cloning the hybridomas, selecting positive clonesthat produce antibodies to the antigen, and isolating the antibodiesfrom the hybridoma cultures. Monoclonal antibodies can be isolated andpurified from hybridoma cultures by a variety of well-establishedtechniques. Such isolation techniques include affinity chromatographywith Protein-A Sepharose, size-exclusion chromatography, andion-exchange chromatography. See, e.g., Coligan et al., sections2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barnes et al., “Purification ofImmunoglobulin G (IgG)” in Methods In Molecular Biology, VOL. 10, pages79-104 (Humana Press 1992).

Antibodies which bind to an invention Glutamate Transporter AssociatedProtein polypeptide can be prepared using an intact polypeptide orfragments containing small peptides of interest as the immunizingantigen. For example, it may be desirable to produce antibodies thatspecifically bind to the amino- or carboxyl-terminal domains of aninvention polypeptide. For the preparation of polyclonal antibodies, thepolypeptide or peptide used to immunize an animal is derived fromtranslated cDNA or chemically synthesized and can be conjugated to acarrier protein, if desired. Commonly used carrier proteins which may bechemically coupled to the immunizing peptide include keyhole limpethemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), tetanustoxoid, and the like.

Invention polyclonal or monoclonal antibodies can be further purified,for example, by binding to and elution from a matrix to which thepolypeptide or a peptide to which the antibodies were raised is bound.Those of skill in the art will know of various techniques common in theimmunology arts for purification and/or concentration of polyclonalantibodies, as well as monoclonal antibodies (See, for example, Coligan,et al., Unit 9, Current Protocols in Immunology, Wiley Interscience,1994, incorporated herein by reference).

The antibodies of the invention can be bound to many different carriersand used to detect the presence of an antigen comprising thepolypeptides of the invention. Examples of well-known carriers includeglass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, agaroses andmagnetite. The nature of the carrier can be either soluble or insolublefor purposes of the invention. Those skilled in the art will know ofother suitable carriers for binding antibodies, or will be able toascertain such, using routine experimentation.

There are many different labels and methods of labeling known to thoseof ordinary skill in the art. Examples of the types of labels which canbe used in the present invention include enzymes, radioisotopes,fluorescent compounds, colloidal metals, chemiluminescent compounds,phosphorescent compounds, and bioluminescent compounds. Those ofordinary skill in the art will know of other suitable labels for bindingto the antibody, or will be able to ascertain such, using routineexperimentation.

Another technique which may also result in greater sensitivity consistsof coupling the antibodies to low molecular weight haptens. Thesehaptens can then be specifically detected by means of a second reaction.For example, it is common to use such haptens as biotin, which reactswith avidin, or dinitrophenyl, puridoxal, and fluorescein, which canreact with specific antihapten antibodies.

In using the monoclonal and polyclonal antibodies of the invention forthe in vivo detection of antigen, e.g., a Glutamate TransporterAssociated Protein, the detectably labeled antibody is given a dosewhich is diagnostically effective. The term “diagnostically effective”means that the amount of detectably labeled antibody is administered insufficient quantity to enable detection of the site having the antigencomprising a polypeptide of the invention for which the antibodies arespecific.

The concentration of detectably labeled antibody which is administeredshould be sufficient such that the binding to those cells having thepolypeptide is detectable compared to the background. Further, it isdesirable that the detectably labeled antibody be rapidly cleared fromthe circulatory system in order to give the best target-to-backgroundsignal ratio.

As a rule, the dosage of detectably labeled antibody for in vivotreatment or diagnosis will vary depending on such factors as age, sex,and extent of disease of the individual. Such dosages may vary, forexample, depending on whether multiple injections are given, antigenicburden, and other factors known to those of skill in the art.

A polynucleotide agent can be contained in a vector, which canfacilitate manipulation of the polynucleotide, including introduction ofthe polynucleotide into a target cell. The vector can be a cloningvector, which is useful for maintaining the polynucleotide, or can be anexpression vector, which contains, in addition to the polynucleotide,regulatory elements useful for expressing the polynucleotide and, wherethe polynucleotide encodes a peptide, for expressing the encoded peptidein a particular cell. An expression vector can contain the expressionelements necessary to achieve, for example, sustained transcription ofthe encoding polynucleotide, or the regulatory elements can beoperatively linked to the polynucleotide prior to its being cloned intothe vector.

An expression vector (or the polynucleotide) generally contains orencodes a promoter sequence, which can provide constitutive or, ifdesired, inducible or tissue specific or developmental stage specificexpression of the encoding polynucleotide, a poly-A recognitionsequence, and a ribosome recognition site or internal ribosome entrysite, or other regulatory elements such as an enhancer, which can betissue specific. The vector also can contain elements required forreplication in a prokaryotic or eukaryotic host system or both, asdesired. Such vectors, which include plasmid vectors and viral vectorssuch as bacteriophage, baculovirus, retrovirus, lentivirus, adenovirus,vaccinia virus, semliki forest virus and adeno-associated virus vectors,are well known and can be purchased from a commercial source (Promega,Madison Wis.; Stratagene, La Jolla Calif.; GIBCO/BRL, Gaithersburg Md.)or can be constructed by one skilled in the art (see, for example, Meth.Enzymol., Vol. 185, Goeddel, ed. (Academic Press, Inc., 1990); Jolly,Canc. Gene Ther. 1:51-64, 1994; Flotte, J. Bioenerg. Biomemb. 25:37-42,1993; Kirshenbaum et al., J. Clin. Invest. 92:381-387, 1993; each ofwhich is incorporated herein by reference).

A polynucleotide useful in a method of the invention also can beoperatively linked to tissue specific regulatory element, for example, aneuron specific regulatory element, such that expression of an encodedpeptide agent is restricted to neurons in an individual, or to neuronsin a mixed population of cells in culture, for example, an organculture. For example, neuronal or glial promoters such as the myelinbasic protein promoter, other neuronal-specific promoters, andastroglial promoters (e.g. GFAP—glial fibrillary acidic protein), knownto those of skill in the art may be used. Muscle-regulatory elementsincluding, for example, the muscle creatine kinase promoter (Sternberget al., Mol. Cell. Biol. 8:2896-2909, 1988, which is incorporated hereinby reference) and the myosin light chain enhancer/promoter (Donoghue etal., Proc. Natl. Acad. Sci., USA 88:5847-5851, 1991, which isincorporated herein by reference) are well known in the art. A varietyof other promoters have been identified which are suitable for upregulating expression in cardiac tissue. Included, for example, are thecardiac I-myosin heavy chain (AMHC) promoter and the cardiac I-actinpromoter. Other examples of tissue-specific regulatory elements include,tissue-specific promoters, pancreatic (insulin or elastase), and actinpromoter in smooth muscle cells. Through the use of promoters, such asmilk-specific promoters, recombinant retroviruses may be isolateddirectly from the biological fluid of the progeny.

A Glutamate Transporter Associated Protein polynucleotide of theinvention can be inserted into a vector, which can be a cloning vectoror a recombinant expression vector. The term “expression vector” refersto a plasmid, virus or other vehicle known in the art that has beenmanipulated by insertion or incorporation of a polynucleotide,particularly, with respect to the present invention, a polynucleotideencoding all or a peptide portion of a Glutamate Transporter AssociatedProtein. Such expression vectors contain a promoter sequence, whichfacilitates the efficient transcription of the inserted genetic sequenceof the host. The expression vector generally contains an origin ofreplication, a promoter, as well as specific genes which allowphenotypic selection of the transformed cells. Vectors suitable for usein the present invention include, but are not limited to, the T7-basedexpression vector for expression in bacteria (Rosenberg, et al., Gene56:125, 1987), the pMSXND expression vector for expression in mammaliancells (Lee and Nathans, J. Biol. Chem. 263:3521, 1988) andbaculovirus-derived vectors for expression in insect cells. The DNAsegment can be present in the vector operably linked to regulatoryelements, for example, a promoter, which can be a T7 promoter,metallothionein I promoter, polyhedrin promoter, or other promoter asdesired, particularly tissue specific promoters or inducible promoters.

Viral expression vectors can be particularly useful for introducing apolynucleotide useful in a method of the invention into a cell,particularly a cell in a subject. Viral vectors provide the advantagethat they can infect host cells with relatively high efficiency and caninfect specific cell types. For example, a polynucleotide encoding aGlutamate Transporter Associated Protein or functional peptide portionthereof can be cloned into a baculovirus vector, which then can be usedto infect an insect host cell, thereby providing a means to producelarge amounts of the encoded protein or peptide portion. The viralvector also can be derived from a virus that infects cells of anorganism of interest, for example, vertebrate host cells such asmammalian, avian or piscine host cells. Viral vectors can beparticularly useful for introducing a polynucleotide useful inperforming a method of the invention into a target cell. Viral vectorshave been developed for use in particular host systems, particularlymammalian systems and include, for example, retroviral vectors, otherlentivirus vectors such as those based on the human immunodeficiencyvirus (HIV), adenovirus vectors, adeno-associated virus vectors,herpesvirus vectors, vaccinia virus vectors, and the like (see Millerand Rosman, BioTechnigues 7:980-990, 1992; Anderson et al., Nature392:25-30 Suppl., 1998; Verma and Somia, Nature 389:239-242, 1997;Wilson, New Engl. J. Med. 334:1185-1187 (1996), each of which isincorporated herein by reference).

When retroviruses, for example, are used for gene transfer, replicationcompetent retroviruses theoretically can develop due to recombination ofretroviral vector and viral gene sequences in the packaging cell lineutilized to produce the retroviral vector. Packaging cell lines in whichthe production of replication competent virus by recombination has beenreduced or eliminated can be used to minimize the likelihood that areplication competent retrovirus will be produced. All retroviral vectorsupernatants used to infect cells are screened for replication competentvirus by standard assays such as PCR and reverse transcriptase assays.Retroviral vectors allow for integration of a heterologous gene into ahost cell genome, which allows for the gene to be passed to daughtercells following cell division.

A polynucleotide, which can be contained in a vector, can be introducedinto a cell by any of a variety of methods known in the art (Sambrook etal., Molecular Cloning: A laboratory manual (Cold Spring HarborLaboratory Press 1989); Ausubel et al., Current Protocols in MolecularBiology, John Wiley and Sons, Baltimore, Md. (1987, and supplementsthrough 1995), each of which is incorporated herein by reference). Suchmethods include, for example, transfection, lipofection, microinjection,electroporation and, with viral vectors, infection; and can include theuse of liposomes, microemulsions or the like, which can facilitateintroduction of the polynucleotide into the cell and can protect thepolynucleotide from degradation prior to its introduction into the cell.The selection of a particular method will depend, for example, on thecell into which the polynucleotide is to be introduced, as well aswhether the cell is isolated in culture, or is in a tissue or organ inculture or in situ.

Introduction of a polynucleotide into a cell by infection with a viralvector is particularly advantageous in that it can efficiently introducethe nucleic acid molecule into a cell ex vivo or in vivo (see, forexample, U.S. Pat. No. 5,399,346, which is incorporated herein byreference). Moreover, viruses are very specialized and can be selectedas vectors based on an ability to infect and propagate in one or a fewspecific cell types. Thus, their natural specificity can be used totarget the nucleic acid molecule contained in the vector to specificcell types. As such, a vector based on an HIV can be used to infect Tcells, a vector based on an adenovirus can be used, for example, toinfect respiratory epithelial cells, a vector based on a herpesvirus canbe used to infect neuronal cells, and the like. Other vectors, such asadeno-associated viruses can have greater host cell range and,therefore, can be used to infect various cell types, although viral ornon-viral vectors also can be modified with specific receptors orligands to alter target specificity through receptor mediated events.

A polynucleotide sequence encoding a Glutamate Transporter AssociatedProtein can be expressed in either prokaryotes or eukaryotes. Hosts caninclude microbial, yeast, insect and mammalian organisms. Methods ofexpressing polynucleotides having eukaryotic or viral sequences inprokaryotes are well known in the art, as are biologically functionalviral and plasmid DNA vectors capable of expression and replication in ahost. Methods for constructing an expression vector containing apolynucleotide of the invention are well known, as are factors to beconsidered in selecting transcriptional or translational controlsignals, including, for example, whether the polynucleotide is to beexpressed preferentially in a particular cell type or under particularconditions (see, for example, Sambrook et al., supra, 1989).

A variety of host cell/expression vector systems can be utilized toexpress a Glutamate Transporter Associated Protein coding sequence,including, but not limited to, microorganisms such as bacteriatransformed with recombinant bacteriophage DNA, plasmid DNA or cosmidDNA expression vectors; yeast cells transformed with recombinant yeastexpression vectors; plant cell systems infected with recombinant virusexpression vectors such as a cauliflower mosaic virus or tobacco mosaicvirus, or transformed with recombinant plasmid expression vector such asa Ti plasmid; insect cells infected with recombinant virus expressionvectors such as a baculovirus; animal cell systems infected withrecombinant virus expression vectors such as a retrovirus, adenovirus orvaccinia virus vector; and transformed animal cell systems geneticallyengineered for stable expression. Where the expressed GlutamateTransporter Associated Protein is post-translationally modified, forexample, by glycosylation, it can be particularly advantageous to selecta host cell/expression vector system that can effect the desiredmodification, for example, a mammalian host cell/expression vectorsystem.

Depending on the host cell/vector system utilized, any of a number ofsuitable transcription and translation elements, including constitutiveand inducible promoters, transcription enhancer elements, transcriptionterminators, and the like can be used in the expression vector (Bitteret al., Meth. Enzymol. 153:516-544, 1987). For example, when cloning inbacterial systems, inducible promoters such as pL of bacteriophage Σ,plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like can be used.When cloning in mammalian cell systems, promoters derived from thegenome of mammalian cells, for example, a human or mouse metallothioneinpromoter, or from mammalian viruses, for example, a retrovirus longterminal repeat, an adenovirus late promoter or a vaccinia virus 7.5Kpromoter, can be used. Promoters produced by recombinant DNA orsynthetic techniques can also be used to provide for transcription ofthe inserted GDF receptors coding sequence.

In yeast cells, a number of vectors containing constitutive or induciblepromoters can be used (see Ausubel et al., supra, 1987, see chapter 13;Grant et al., Meth. Enzymol. 153:516-544, 1987; Glover, DNA Cloning Vol.II (IRL Press, 1986), see chapter 3; Bitter, Meth. Enzymol. 152:673-684,1987; see, also, The Molecular Biology of the Yeast Saccharomyces (Eds.,Strathern et al., Cold Spring Harbor Laboratory Press, 1982), Vols. Iand II). A constitutive yeast promoter such as ADH or LEU2 or aninducible promoter such as GAL can be used (Rothstein, DNA Cloning Vol.II (supra, 1986), chapter 3). Alternatively, vectors can be used whichpromote integration of foreign DNA sequences into the yeast chromosome.

Eukaryotic systems, particularly mammalian expression systems, allow forproper post-translational modifications of expressed mammalian proteins.Eukaryotic cells which possess the cellular machinery for properprocessing of the primary transcript, glycosylation, phosphorylation,and advantageously, plasma membrane insertion of the gene product can beused as host cells for the expression of a Glutamate TransporterAssociated Protein, or functional peptide portion thereof.

Mammalian cell systems which utilize recombinant viruses or viralelements to direct expression can be engineered. For example, when usingadenovirus expression vectors, the Glutamate Transporter AssociatedProtein coding sequence can be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. Alternatively, the vaccinia virus 7.5Kpromoter can be used (Mackett et al., Proc. Natl. Acad. Sci., USA79:7415-7419, 1982; Mackett et al., J. Virol. 49:857-864, 1984; Panicaliet al., Proc. Natl. Acad. Sci., USA 79:4927-4931, 1982). Particularlyuseful are bovine papilloma virus vectors, which can replicate asextrachromosomal elements (Sarver et al., Mol. Cell. Biol. 1:486, 1981).Shortly after entry of this DNA into mouse cells, the plasmid replicatesto about 100 to 200 copies per cell. Transcription of the inserted cDNAdoes not require integration of the plasmid into the host cellchromosome, thereby yielding a high level of expression. These vectorscan be used for stable expression by including a selectable marker inthe plasmid, such as, for example, the neo gene. Alternatively, theretroviral genome can be modified for use as a vector capable ofintroducing and directing the expression of the Glutamate TransporterAssociated Protein gene in host cells (Cone and Mulligan, Proc. Natl.Acad. Sci., USA 81:6349-6353, 1984). High level expression can also beachieved using inducible promoters, including, but not limited to, themetallothionein IIA promoter and heat shock promoters.

For long term, high yield production of recombinant proteins, stableexpression is preferred. Rather than using expression vectors whichcontain viral origins of replication, host cells can be transformed withGlutamate Transporter Associated Protein cDNA controlled by appropriateexpression control elements such as promoter, enhancer, sequences,transcription terminators, and polyadenylation sites, and a selectablemarker. The selectable marker in the recombinant plasmid can conferresistance to the selection, and allows cells to stably integrate theplasmid into their chromosomes and grow to form foci, which, in turn canbe cloned and expanded into cell lines. For example, following theintroduction of foreign DNA, engineered cells can be allowed to grow for1 to 2 days in an enriched media, and then are switched to a selectivemedia. A number of selection systems can be used, including, but notlimited to, the herpes simplex virus thymidine kinase (Wigler et al.,Cell 11:223, 1977), hypoxanthine-guanine phosphoribosyltransferase(Szybalska and Szybalski, Proc. Natl. Acad. Sci., USA 48:2026, 1982),and adenine phosphoribosyltransferase (Lowy, et al., Cell 22:817, 1980)genes can be employed in tk⁻, hgprt⁻ or aprt⁻ cells respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler, et al., Proc.Natl. Acad. Sci. USA 77:3567, 1980; O'Hare et al., Proc. Natl. Acad.Sci., USA 78: 1527, 1981); gpt, which confers resistance to mycophenolicacid (Mulligan and Berg, Proc. Natl. Acad. Sci., USA 78:2072, 1981);neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin et al., J. Mol. Biol. 150:1, 1981); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147, 1984)genes. Additional selectable genes, including trpB, which allows cellsto utilize indole in place of tryptophan; hisD, which allows cells toutilize histinol in place of histidine (Hartman and Mulligan, Proc.Natl. Acad. Sci., USA 85:8047, 1988); and ODC (ornithine decarboxylase)which confers resistance to the omithine decarboxylase inhibitor,2-(difluoromethyl)-DL-omithine, DFMO (McConlogue, Curr. Comm. Mol. Biol.(Cold Spring Harbor Laboratory Press, 1987), also have been described.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors can be used. Eukaryotic cells can also becotransformed with DNA sequences encoding Glutamate TransporterAssociated Proteins of the invention, and a second foreign DNA moleculeencoding a selectable phenotype, such as the herpes simplex thymidinekinase gene. Another method is to use a eukaryotic viral vector, such assimian virus 40 (SV40) or bovine papilloma virus, to transiently infector transform eukaryotic cells and express the protein. (Gluzman,Eukarvotic Viral Vectors (Cold Spring Harbor Laboratory Press, 1982)).

The invention provides a method for producing a polypeptidecharacterized as interacting with a glutamate transporter protein;modulating intracellular glutamate transport; having an expressionpattern in Purkinje cells of the brain; and being hydrophobic. Theinvention also provides a method for producing a polypeptide encoded bythe nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:6 orfragments thereof, including culturing the host cell under conditionssuitable for the expression of the polypeptide and recovering thepolypeptide from the host cell culture.

A Glutamate Transporter Associated Protein polypeptide or a fragmentthereof, can be encoded by a recombinant or non-recombinant nucleic acidmolecule and expressed in a cell. Preparation of a Glutamate TransporterAssociated Protein polypeptide by recombinant methods provides severaladvantages. In particular, the nucleic acid sequence encoding theGlutamate Transporter Associated Protein polypeptide can includeadditional nucleotide sequences encoding, for example, peptides usefulfor recovering the Glutamate Transporter Associated Protein polypeptidefrom the host cell. A Glutamate Transporter Associated Proteinpolypeptide can be recovered using well known methods, including, forexample, precipitation, gel filtration, ion exchange, reverse-phase, oraffinity chromatography (see, for example, Deutscher et al., “Guide toProtein Purification” in Meth. Enzymol., Vol. 182, (Academic Press,1990)). Such methods also can be used to purify a fragment of aGlutamate Transporter Associated Protein polypeptide, for example, aparticular binding sequence, from a cell in which it is naturallyexpressed.

A recombinant nucleic acid molecule encoding a Glutamate TransporterAssociated Protein polypeptide or a fragment thereof can include, forexample, a protease site, which can facilitate cleavage of the GlutamateTransporter Associated Protein polypeptide from a non-GlutamateTransporter Associated Protein polypeptide sequence, for example, a tagpeptide, secretory peptide, or the like. As such, the recombinantnucleic acid molecule also can encode a tag peptide such as apolyhistidine sequence, a FLAG peptide (Hopp et al., Biotechnology6:1204 (1988)), a glutathione S-transferase polypeptide or the like,which can be bound by divalent metal ions, a specific antibody (U.S.Pat. No. 5,011,912), or glutathione, respectively, thus facilitatingrecovery and purification of the Glutamate Transporter AssociatedProtein polypeptide comprising the peptide tag. Such tag peptides alsocan facilitate identification of the Glutamate Transporter AssociatedProtein polypeptide through stages of synthesis, chemical or enzymaticmodification, linkage, or the like. Methods for purifying polypeptidescomprising such tags are well known in the art and the reagents forperforming such methods are commercially available.

A nucleic acid molecule encoding a Glutamate Transporter AssociatedProtein polypeptide can be engineered to contain one or more restrictionendonuclease recognition and cleavage sites, which can facilitate, forexample, substitution of an element of the Glutamate TransporterAssociated Protein polypeptide such as the selective recognition domainor, where present, a spacer element. As such, related GlutamateTransporter Associated Protein polypeptides can be prepared, each havinga similar activity, but having specificity for differentfunction-forming contexts. A restriction endonuclease site also can beengineered into (or out of) the sequence coding a peptide portion of theGlutamate Transporter Associated Protein polypeptide, and can, but neednot change one or more amino acids encoded by the particular sequence.Such a site can provide a simple means to identify the nucleic acidsequence, based on cleavage (or lack of cleavage) following contact withthe relevant restriction endonuclease, and, where introduction of thesite changes an amino acid, can further provide advantages based on thesubstitution.

In another embodiment of the invention there is provided a substantiallypure polypeptide which interacts with amino acid sequence QEAELTLP (SEQID NO:9) or the amino acid sequence GRGGNESVM (SEQ ID NO:10). In apreferred embodiment, polypeptides interact with a Glutamate TransporterAssociated Protein encoded by a polynucleotide that hybridizes to SEQ IDNO: 1. An exemplary protein containing amino acid sequences QEAELTLP(SEQ ID NO:9) and GRGGNESVM (SEQ ID NO: 10) is glutamate transportprotein EAAT4 (see Examples). In another embodiment of the invention,there is provided a polynucleotide encoding a substantially purepolypeptide which interacts with amino acid sequence QEAELTLP (SEQ IDNO:9) or the amino acid sequence GRGGNESVM (SEQ ID NO:10).

Another embodiment of the invention provides a substantially purepolypeptide which interacts with a polypeptide having the sequence ofamino acids found at amino acid residues 527 to 534 of EAAT4 (SEQ IDNO:9). Still another embodiment of the invention provides asubstantially pure polypeptide which interacts with a polypeptidesequence having the sequence of amino acid found at amino acid residues555 to 561 of EAAT4 (SEQ ID NO:10).

Still another embodiment of the invention provides a substantially purepolypeptide which interacts with the amino acid sequence set forth inSEQ ID NO:12. Also provided is a substantially pure polypeptide whichinteracts with the amino acid sequence set forth in SEQ ID NO: 13. Suchamino acid sequence are used as “bait” sequences in yeast two-hybridscreen (See Example 1). Polypeptides identified in such screens areinteracting proteins. Interacting proteins can mediate or modulate theactivities of intracellular proteins.

A method is provided for identifying a compound that modulates acellular response mediated by a Glutamate Transporter AssociatedProtein. The method includes incubating the compound with a cellexpressing a Glutamate Transporter Associated Protein and a glutamatetransporter protein under conditions sufficient to permit the compoundto interact with the cell. The effect of the compound on the cellularresponse is determined, either directly or indirectly, and a cellularresponse is then compared with a cellular response of a control cell. Asuitable control includes, but is not limited to, a cellular response ofa cell not contacted with the compound. The cell may be any cell ofinterest, including but not limited to neuronal cells, glial cells,cardiac cells, bronchial cells, uterine cells, testicular cells, livercells, renal cells, intestinal cells, cells from the thymus and spleen,placental cells, endothelial cells, endocrine cells including thyroid,parathyroid, pituitary and the like, smooth muscle cells and skeletalmuscle cells. The term “incubating” includes conditions which allowcontact between the test compound and the cell of interest. “Contacting”may include in solution or in solid phase.

The cellular response can be an increase in glutamate transport or adecrease in glutamate transport. Glutamate transport can be assessed bymeasuring glutamate uptake assays (see Example 8) and other assays knownin the art.

The cellular response can be an increase in cytoskeletal stability or adecrease in cytoskeletal stability. Cytoskeletal stability can beassessed for example, by examining the formation and maintenance ofintracellular protein interaction, cell-surface receptor clustering,clustering of glutamate transporter proteins, and the like. Methods fordemonstrating such cellular responses are well known in the art (e.g.biochemical methods and histological methods). (See Komau et al. (1997)Curr. Opin. Neurobiol. 7:368-373; and Huganir et al. (2000) Trends inCell Biol. 10:274-280, each of which are herein incorporated byreference in their entirety and Examples section for additionalmethodology).

The cellular response can be an increase in chloride flux or a decreasein chloride flux. Chloride flux can be assessed by methods known tothose of skill in the art such as electrophysiological methodsincluding, but not limited to, patch clamp analysis.

Glutamate Transporter Associated Proteins contemplated for use in theinvention method includes, for example, GTRAP4-41, GTRAP4-48,PCTAIRE-1a, PCTAIRE-1b, and GTRAP3-18. Glutamate transport proteinscontemplated for use in the invention method include GLAST, GLT-1,EAAC1, EAAT1, EAAT2, EAAT3, EAAT4 and EAAT5.

In one preferred embodiment of the invention, the glutamate transportprotein is EATT4 and the Glutamate Transporter Associated Protein isGTRAP4-41, GTRAP4-48, PCTAIRE-1a or PCTAIRE-1b. In another embodiment ofthe invention, the glutamate transport protein is EAAC1 and theGlutamate Transporter Associated Protein is GTRAP3-18.

In an embodiment of the invention, the cell expressing a GlutamateTransporter Associated Protein further expresses a RhoGEF protein. TheRho family of GTP-binding proteins regulates the rearrangement of theactin cytoskeleton. At least one Glutamate Transporter AssociatedProtein has a domain that permits interaction with a a guaninenucleotide exchange factor (GEF).

Compounds which modulate a cellular response include peptides,peptidomimetics, polypeptides, pharmaceuticals, chemical compounds andbiological agents, for example. Antibodies, anti-epileptic compounds andcombinatorial compound libraries can also be tested using the method ofthe invention. One class of organic molecules, preferably small organiccompounds having a molecular weight of more than 50 and less than about2,500 Daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups. Thecandidate agents often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups.

The test agent may also be a combinatorial library for screening aplurality of compounds. Compounds such as peptides identified in themethod of the invention can be further cloned, sequenced, and the like,either in solution of after binding to a solid support, by any methodusually applied to the isolation of a specific DNA sequence Moleculartechniques for DNA analysis (Landegren et al., Science 242:229-237,1988) and cloning have been reviewed (Sambrook et al., MolecularCloning: a Laboratory Manual, 2nd Ed.; Cold Spring Harbor LaboratoryPress, Plainview, N.Y., 1998, herein incorporated by reference).

Candidate compounds are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc., to producestructural analogs. Candidate agents are also found among biomoleculesincluding, but not limited to: peptides, saccharides, fatty acids,steroids, purines, pyrimidines, derivatives, structural analogs orcombinations thereof.

A variety of other agents may be included in the screening assay. Theseinclude agents like salts, neutral proteins, e.g., albumin, detergents,etc. that are used to facilitate optimal protein-protein binding and/orreduce nonspecific or background interactions. Reagents that improve theefficiency of the assay, such as protease inhibitors, nucleaseinhibitors, antimicrobial agents and the like may be used. The mixtureof components are added in any order that provides for the requisitebinding. Incubations are performed at any suitable temperature,typically between 4 and 40° C. Incubation periods are selected foroptimum activity, but may also be optimized to facilitate rapidhigh-throughput screening. Typically between 0.1 and 10 h will besufficient.

Another embodiment of the invention provides a method for identifying acompound that can inhibit an interaction between a Glutamate TransporterAssociated Protein and a glutamate transporter protein. The methodincludes contacting a Glutamate Transporter Associated Protein with aglutamate transporter protein in the presence of the compound, andcomparing the formation of a Glutamate Transporter AssociatedProtein-glutamate transporter complex in the presence of the compoundwith the formation of the complex in the absence of the compound.Compounds that affect complex formation include peptides, polypeptides,pepidomimetics, chemical compounds and biological agents.

Contacting includes in solution and solid phase. In a preferredembodiment, isolated Glutamate Associated Transporter Proteins areutilized. However, partially purified proteins, fractions of cellextracts, whole cell extracts, or intact cells may be utilized with themethod of the invention.

The complex of the Glutamate Associated Transporter Protein and theglutamate transporter protein can be separated from uncomplexedcomponents by conventional means, well known to one of skill in the art.Separation can be accomplished by size separation, physical separation,antibody-mediated separation, or other standard methods. For example,immunoprecipitation or gel electrophoresis can be used to separateGlutamate Transporter Associated Protein-glutamate transporter proteincomplex from components that are not part of the complex (See Examplessection for details).

Also provided is a method of modulating glutamate transport in a subjectin need thereof. The method includes administering to the subject atherapeutically effective amount of a compound that modulates expressionor activity of a Glutamate Transporter Associated Protein, therebymodulating glutamate transport.

A method is further provided for treating a subject with a disorderassociated with glutamate transport comprising administering to thesubject a therapeutically effective amount of a compound that modulatesGlutamate Transporter Associated Protein activity or interaction withglutamate transporter protein.

Essentially, any disorder that is etiologically linked to glutamatetransport or to a Glutamate Transporter Associated Protein could beconsidered susceptible to treatment with an agent that modulatesGlutamate Transporter Associated Protein activity. The disorder may be aneuronal cell disorder. Examples of neuronal cell disorders include butare not limited to epilepsy, neurodegenerative disease (e.g. Alzheimer'sdisease, Huntington's disease, Amyotrophic lateral sclerosis,Parkinson's disease), spinocerebellar ataxia (SCA), especially of theSCA type 1, multiple sclerosis, disorders of neurotransmittermetabolism, including GABA metabolism and the like, Alzheimer's disease,Parkinson's disease, stroke, and brain or spinal cord injury/damage,including ischemic injury, and the like. Disorders also includeglutamate toxicity, a disorder of memory, a disorder of learning or adisorder of brain development, and the like. Also included are disordersof glutamate-GABA imbalance such as schizophrenia, and the like.

In a preferred embodiment, the Glutamate Transporter Associated Proteinis GTAP4-4 1, GTRAP4-48 or PCTAIRE- 1 (including PCTAIRE-1a andPCTAIRE-1b) and the disorder is a disorder of the nervous system such asneurodegeneration or spinocerebellar ataxia type 1.

When the Glutamate Transporter Associated Protein is GTRAP3-18 thedisorder is epilepsy or a disorder of GABA metabolism (e.g. tremors,spasticity, schizophrenia), for example.

Treatment can include modulation of Glutamate Transporter AssociatedProtein expression or activity by administration of a therapeuticallyeffective amount of a compound that modulates Glutamate TransporterAssociated Protein or Glutamate Transporter Associated Protein activity.The term “modulate” envisions the suppression of Glutamate TransporterAssociated Protein activity or expression when the Glutamate TransporterAssociated Protein is overexpressed or has an increased activity ascompared to a control. The term “modulate” also includes theaugmentation of the expression of Glutamate Transporter AssociatedProtein when it is underexpressed or has a decreased activity ascompared to a control. The term “compound” as used herein describes anymolecule, e.g., protein, nucleic acid, or pharmaceutical, with thecapability of altering the expression of Glutamate TransporterAssociated Protein polynucleotide or activity of Glutamate TransporterAssociated Protein. Treatment can inhibit the transcription ortranslation of a Glutamate Transporter Associated Protein nucleotidesequence, inhibit the interaction of a domain of Glutamate TransporterAssociated Protein with its target protein, may increase the avidity ofthis interaction by means of allosteric effects, may block the bindingactivity of a domain of Glutamate Transporter Associated Protein orinfluence other functional properties of Glutamate TransporterAssociated Proteins.

Candidate agents include nucleic acids that interfere with expression ofGlutamate Transporter Associated Protein, such as an antisense nucleicacid, ribozymes, and the like. Candidate agents also encompass numerouschemical classes wherein the agent modulates Glutamate TransporterAssociated Protein expression or activity. For example, when theGlutamate Transporter Associated Protein is GTRAP3-18, the compound canbe a polynucleotide having a nucleic acid sequence substantially similarto SEQ ID NO:20 (5′-GAGCGGGGCAAGGTTCAC-3′). A nucleotide encoded by SEQID NO:20 is antisense to the nucleic acid sequence of GTRRAP3-18 (SeeExample 13). GTRAP3-18 can also be modulated by retinoic acid (SeeExample 14).

When the Glutamate Transporter Associated Protein is GTRAP4-41,GTRAP4-48 or PCTAIRE-1, modulatory compounds include a polynucleotidehaving a nucleic acid sequence that is substantially similar to anantisense nucleic acid sequence that binds to a polynucleotide encodingGTRAP4-41, GTRAP4-48 or PCTAIRE-1.

Modulation of glutamate transport can be an increase in glutamatetransport or a decrease in glutamate transport. When a disorder isassociated with an increase in glutamate transport, compounds thatdecrease glutamate transport can be used. For example, compounds thatmodulate expression of GTRAP3-18 are contemplated. When a disorder isassociated with a decrease in glutamate transport, compound thatincrease glutamate transport are contemplated. For example, compoundsthat modulate expression of GTRAP4-41, GTRAP4-48, or PCTAIRE-1 (a and b)are contemplated.

Detection of altered (decreased or increased) levels of GlutamateTransporter Associated Protein expression can be accomplished byhybridization of nucleic acids isolated from a cell of interest with aGlutamate Transporter Associated Protein of the invention. Analysis,such as Northern Blot analysis, are utilized to quantitate expression ofGlutamate Transporter Associated Protein, such as to measure GlutamateTransporter Associated Protein transcripts. Other standard nucleic aciddetection techniques will be known to those of skill in the art.Detection of altered levels of Glutamate Transporter Associated Proteincan also accomplished using assays designed to detect GlutamateTransporter Associated Protein polypeptide. For example, antibodies orpeptides that specifically bind a Glutamate Transporter AssociatedProtein polypeptide can be utilized. Analyses, such as radioimmune assayor immunohistochemistry, are then used to measure Glutamate TransporterAssociated Protein, such as to measure protein concentrationqualitatively or quantitatively.

Where a disorder is associated with the increased expression ofGlutamate Transporter Associated Protein, nucleic acid sequences thatinterfere with the expression of Glutamate Transporter AssociatedProtein can be used. This approach also utilizes, for example, antisensenucleic acid, ribozymes, or triplex agents to block transcription ortranslation of Glutamate Transporter Associated Protein mRNA, either bymasking that mRNA with an antisense nucleic acid or triplex agent, or bycleaving it with a ribozyme in disorders associated with increasedGlutamate Transporter Associated Protein. Alternatively, a dominantnegative form of Glutamate Transporter Associated Protein polypeptidecould be administered.

When Glutamate Transporter Associated Protein is overexpressed,candidate agents include antisense nucleic acid sequences. Antisensenucleic acids are DNA or RNA molecules that are complementary to atleast a portion of a specific mRNA molecule (Weintraub, 1990, ScientificAmerican, 262:40). In the cell, the antisense nucleic acids hybridize tothe corresponding mRNA, forming a double-stranded molecule. Theantisense nucleic acids interfere with the translation of the mRNA,since the cell will not translate a mRNA that is double-stranded.Antisense oligomers of about 15 nucleotides are preferred, since theyare easily synthesized and are less likely to cause problems than largermolecules when introduced into the target cell. The use of antisensemethods to inhibit the in vitro translation of genes is well known inthe art (Marcus-Sakura, 1988, Anal. Biochem., 172:289).

Use of an oligonucleotide to stall transcription is known as the triplexstrategy since the oligomer winds around double-helical DNA, forming athree-strand helix. Therefore, these triplex compounds can be designedto recognize a unique site on a chosen gene (Maher, et al., 1991,Antisense Res. and Dev., 1(3):227; Helene, C., 1991, Anticancer DrugDesign, 6:569).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single-stranded RNA in a manner analogous to DNArestriction endonucleases. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech, 1988, J. Amer. Med. Assn., 260:3030). A major advantageof this approach is that, because they are sequence-specific, only mRNAswith particular sequences are inactivated.

There are two basic types of ribozymes namely, tetrahymena-type(Hasselhoff, 1988, Nature, 334:585) and “hammerhead”-type.Tetrahymena-type ribozymes recognize sequences which are four bases inlength, while “hammerhead”-type ribozymes recognize base sequences 11-18bases in length. The longer the recognition sequence, the greater thelikelihood that the sequence will occur exclusively in the target mRNAspecies. Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating a specific mRNA species and18-based recognition sequences are preferable to shorter recognitionsequences.

When a disorder is associated with the decreased expression of GlutamateTransporter Associated Protein, nucleic acid sequences that encodeGlutamate Transporter Associated Protein can be used. An agent whichmodulates Glutamate Transporter Associated Protein expression includes apolynucleotide encoding a polypeptide of SEQ ID NO:2, SEQ ID NO:4, SEQID NO:6, SEQ ID NO:8, SEQ ID NO:22, or a conservative variant thereof.Alternatively, an agent of use with the subject invention includesagents that increase the expression of a polynucleotide encodingGlutamate Transporter Associated Protein or an agent that increases theactivity of Glutamate Transporter Associated Protein polypeptide.

In another series of embodiments, the present invention providestransgenic animal models diseases or disorders associated with mutationsin the Glutamate Transporter Associated Protein genes. The animal may beessentially any amphibian, reptile, fish, mammal, and the like.Preferably, the transgenic animal is mammalian including rats, mice,hamsters, guinea pigs, rabbits, dogs, cats, goats, sheep, pigs, andnon-human primates. In addition, invertebrate models, includingnematodes and insects, may be used for certain applications. The animalmodels are produced by standard transgenic methods includingmicroinjection, transfection, or by other forms of transformation ofembryonic stem cells, zygotes, gametes, and germ line cells with vectorsincluding genomic or cDNA fragments, minigenes, homologous recombinationvectors, viral insertion vectors and the like. Suitable vectors includevaccinia virus, adenovirus, adeno associated virus, retrovirus, liposometransport, neuraltropic viruses, Herpes simplex virus, and the like. Theanimal models may include transgenic sequences comprising or derivedfrom Glutamate Transporter Associated Proteins including normal andmutant sequences, intronic, exonic and untranslated sequences, andsequences encoding subsets of Glutamate Transporter Associated Proteinsuch as functional domains.

The major types of animal models provided include: (1) Animals in whicha normal human Glutamate Transporter Associated Protein gene has beenrecombinantly introduced into the genome of the animal as an additionalgene, under the regulation of either an exogenous or an endogenouspromoter element, and as either a minigene or a large genomic fragment;in which a normal human Glutamate Transporter Associated Protein genehas been recombinantly substituted for one or both copies of theanimal's homologous Glutamate Transporter Associated Protein gene byhomologous recombination or gene targeting; and/or in which one or bothcopies of one of the animal's homologous Glutamate TransporterAssociated Protein genes have been recombinantly “humanized” by thepartial substitution of sequences encoding the human homologue byhomologous recombination or gene targeting. (2) Animals in which amutant human Glutamate Transporter Associated Protein gene has beenrecombinantly introduced into the genome of the animal as an additionalgene, under the regulation of either an exogenous or an endogenouspromoter element, and as either a minigene or a large genomic fragment;in which a mutant human Glutamate Transporter Associated Protein genehas been recombinantly substituted for one or both copies of theanimal's homologous Glutamate Transporter Associated Protein gene byhomologous recombination or gene targeting; and/or in which one or bothcopies of one of the animal's homologous Glutamate TransporterAssociated Protein genes have been recombinantly “humanized” by thepartial substitution of sequences encoding a mutant human homologue byhomologous recombination or gene targeting. (3) Animals in which amutant version of one of that animal's Glutamate Transporter AssociatedProtein genes has been recombinantly introduced into the genome of theanimal as an additional gene, under the regulation of either anexogenous or an endogenous promoter element, and as either a minigene ora large genomic fragment; and/or in which a mutant version of one ofthat animal's Glutamate Transporter Associated Protein genes has beenrecombinantly substituted for one or both copies of the animal'shomologous Glutamate Transporter Associated Protein gene by homologousrecombination or gene targeting. (4) “Knock-out” animals in which one orboth copies of one of the animal's Glutamate Transporter AssociatedProtein genes have been partially or completely deleted by homologousrecombination or gene targeting, or have been inactivated by theinsertion or substitution by homologous recombination or gene targetingof exogenous sequences.

In a preferred embodiment of the invention, there is provided atransgenic non-human animal having a transgene that expresses aGlutamate Transporter Associated Protein-encoding polynucleotidechromosomally integrated into the germ cells of the animal. Animals arereferred to as “transgenic” when such animal has had a heterologous DNAsequence, or one or more additional DNA sequences normally endogenous tothe animal (collectively referred to herein as “transgenes”)chromosomally integrated into the germ cells of the animal. Thetransgenic animal (including its progeny) will also have the transgenefortuitously integrated into the chromosomes of somatic cells.

Various methods to make the transgenic animals of the subject inventioncan be employed. Generally speaking, three such methods may be employed.In one such method, an embryo at the pronuclear stage (a “one cellembryo”) is harvested from a female and the transgene is microinjectedinto the embryo, in which case the transgene will be chromosomallyintegrated into both the germ cells and somatic cells of the resultingmature animal. In another such method, embryonic stem cells are isolatedand the transgene incorporated therein by electroporation, plasmidtransfection or microinjection, followed by reintroduction of the stemcells into the embryo where they colonize and contribute to the germline. Methods for microinjection of mammalian species is described inU.S. Pat. No. 4,873,191. In yet another such method, embryonic cells areinfected with a retrovirus containing the transgene whereby the germcells of the embryo have the transgene chromosomally integrated therein.When the animals to be made transgenic are avian, because avianfertilized ova generally go through cell division for the first twentyhours in the oviduct, microinjection into the pronucleus of thefertilized egg is problematic due to the inaccessibility of thepronucleus. Therefore, of the methods to make transgenic animalsdescribed generally above, retrovirus infection is preferred for avianspecies, for example as described in U.S. Pat. No. 5,162,215. Ifmicroinjection is to be used with avian species, however, a recentlypublished procedure by Love et al., (Biotechnology, 12, Jan 1994) can beutilized whereby the embryo is obtained from a sacrificed henapproximately two and one-half h after the laying of the previous laidegg, the transgene is microinjected into the cytoplasm of the germinaldisc and the embryo is cultured in a host shell until maturity. When theanimals to be made transgenic are bovine or porcine, microinjection canbe hampered by the opacity of the ova thereby making the nucleidifficult to identify by traditional differential interference-contrastmicroscopy. To overcome this problem, the ova can first be centrifugedto segregate the pronuclei for better visualization.

The non-human animals of the invention are murine typically (e.g.,mouse). The transgenic non-human animals of the invention are producedby introducing “transgenes” into the germline of the non-human animal.Embryonal target cells at various developmental stages can be used tointroduce transgenes. Different methods are used depending on the stageof development of the embryonal target cell. The zygote is the besttarget for microinjection. The use of zygotes as a target for genetransfer has a major advantage in that in most cases the injected DNAwill be incorporated into the host gene before the first cleavage(Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438-4442, 1985). As aconsequence, all cells of the transgenic non-human animal will carry theincorporated transgene. This will in general also be reflected in theefficient transmission of the transgene to offspring of the foundersince 50% of the germ cells will harbor the transgene.

The term “transgenic” is used to describe an animal which includesexogenous genetic material within all of its cells. A “transgenic”animal can be produced by cross-breeding two chimeric animals whichinclude exogenous genetic material within cells used in reproduction.Twenty-five percent of the resulting offspring will be transgenic i.e.,animals which include the exogenous genetic material within all of theircells in both alleles. Fifty percent of the resulting animals willinclude the exogenous genetic material within one allele and twenty fivepercent will include no exogenous genetic material.

In the microinjection method useful in the practice of the subjectinvention, the transgene is digested and purified free from any vectorDNA e.g. by gel electrophoresis. It is preferred that the transgeneinclude an operatively associated promoter which interacts with cellularproteins involved in transcription, ultimately resulting in constitutiveexpression. Promoters useful in this regard include those fromcytomegalovirus (CMV), Moloney leukemia virus (MLV), and herpes virus,as well as those from the genes encoding metallothionine, skeletalactin, P-enolpyruvate carboxylase (PEPCK), phosphoglycerate (PGK), DHFR,and thymidine kinase. Promoters for viral long terminal repeats (LTRs)such as Rous Sarcoma Virus can also be employed. Constructs useful inplasmid transfection of embryonic stem cells will employ additionalregulatory elements well known in the art such as enhancer elements tostimulate transcription, splice acceptors, termination andpolyadenylation signals, and ribosome binding sites to permittranslation.

Retroviral infection can also be used to introduce transgene into anon-human animal, as described above. The developing non-human embryocan be cultured in vitro to the blastocyst stage. During this time, theblastomeres can be targets for retro viral infection (Jaenich, R., Proc.Natl. Acad. Sci USA 73:1260-1264, 1976). Efficient infection of theblastomeres is obtained by enzymatic treatment to remove the zonapellucida (Hogan, et al. (1986) in Manipulating the Mouse Embryo, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The viralvector system used to introduce the transgene is typically areplication-defective retro virus carrying the transgene (Jahner, etal., Proc. Natl. Acad. Sci. USA 82:6927-6931, 1985; Van der Putten, etal., Proc. Natl. Acad. Sci USA 82:6148-6152, 1985). Transfection iseasily and efficiently obtained by culturing the blastomeres on amonolayer of virus-producing cells (Van der Putten, supra; Stewart, etal., EMBO J 6:383-388, 1987). Alternatively, infection can be performedat a later stage. Virus or virus-producing cells can be injected intothe blastocoel (D. Jahner et al., Nature 298:623-628, 1982). Most of thefounders will be mosaic for the transgene since incorporation occursonly in a subset of the cells which formed the transgenic nonhumananimal. Further, the founder may contain various retro viral insertionsof the transgene at different positions in the genome which generallywill segregate in the offspring. In addition, it is also possible tointroduce transgenes into the germ line, albeit with low efficiency, byintrauterine retroviral infection of the midgestation embryo (D. Jahneret al., supra).

A third type of target cell for transgene introduction is the embryonalstem cell (ES). ES cells are obtained from pre-implantation embryoscultured in vitro and fused with embryos (M. J. Evans et al. Nature292:154-156, 1981; M. O. Bradley et al., Nature 309: 255-258, 1984;Gossler, et al., Proc. Natl. Acad. Sci USA 83: 9065-9069, 1986; andRobertson et al., Nature 322:445-448, 1986). Transgenes can beefficiently introduced into the ES cells by DNA transfection or by retrovirus-mediated transduction. Such transformed ES cells can thereafter becombined with blastocysts from a nonhuman animal. The ES cellsthereafter colonize the embryo and contribute to the germ line of theresulting chimeric animal. (For review see Jaenisch, R., Science 240:1468-1474, 1988).

“Transformed” means a cell into which (or into an ancestor of which) hasbeen introduced, by means of recombinant nucleic acid techniques, aheterologous nucleic acid molecule. “Heterologous” refers to a nucleicacid sequence that either originates from another species or is modifiedfrom either its original form or the form primarily expressed in thecell.

“Transgene” means any piece of DNA which is inserted by artifice into acell, and becomes part of the genome of the organism (i.e., eitherstably integrated or as a stable extrachromosomal element) whichdevelops from that cell. Such a transgene may include a gene which ispartly or entirely heterologous (i.e., foreign) to the transgenicorganism, or may represent a gene homologous to an endogenous gene ofthe organism. Included within this definition is a transgene created bythe providing of an RNA sequence which is transcribed into DNA and thenincorporated into the genome. The transgenes of the invention includeDNA sequences which encode Glutamate Transporter Associated Proteinpolypeptide-sense and antisense polynucleotides, which may be expressedin a transgenic non-human animal. The term “transgenic” as used hereinadditionally includes any organism whose genome has been altered by invitro manipulation of the early embryo or fertilized egg or by anytransgenic technology to induce a specific gene knockout. As usedherein, the term “transgenic” includes any transgenic technologyfamiliar to those in the art which can produce an organism carrying anintroduced transgene or one in which an endogenous gene has beenrendered non-functional or “knocked out”.

Another embodiment of the invention provides a computer readable mediumhaving store thereon a nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 andsequences substantially identical thereto, or a polypeptide sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:22 and sequences substantially identicalthereto.

A further embodiment of the invention provides a computer systemcomprising a processor and a data storage device wherein said datestorage device has stored thereon a nucleic acid sequence selected fromthe group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7 and sequences substantially identical thereto, or a polypeptidesequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:22 and sequences substantiallyidentical thereto. The computer system, additionally can contain asequence comparison algorithm and a data storage device having at leastone reference sequence stored on it. The sequence comparison algorithmcomprises a computer program which indicates polymorphisms. The term“polymorphism”, as used herein, refers to the existence of multiplealleles at a single locus. Polymorphism can be are several typesincluding, for example, those that change DNA sequence but do not changeprotein sequence, those that change protein sequence without changingfunction, those that create proteins with a different activity, andthose that create proteins that are non-functional.

Embodiments of the invention include systems (e.g., internet basedsystems), particularly computer systems which store and manipulate thecoordinate and sequence information described herein. One example of acomputer system 100 is illustrated in block diagram form in FIG. 10. Asused herein, “a computer system” refers to the hardware components,software components, and data storage components used to analyze thecoordinates and sequences as set forth herein. The computer system 100typically includes a processor for processing, accessing andmanipulating the sequence data. The processor 105 can be any well-knowntype of central processing unit, such as, for example, the Pentium IIIfrom Intel Corporation, or similar processor from Sun, Motorola, Compaq,AMD or International Business Machines.

Typically the computer system 100 is a general purpose system thatcomprises the processor 105 and one or more internal data storagecomponents 110 for storing data, and one or more data retrieving devicesfor retrieving the data stored on the data storage components. A skilledartisan can readily appreciate that any one of the currently availablecomputer systems are suitable.

In one particular embodiment, the computer system 100 includes aprocessor 105 connected to a bus which is connected to a main memory 115(preferably implemented as RAM) and one or more internal data storagedevices 110, such as a hard drive and/or other computer readable mediahaving data recorded thereon. In some embodiments, the computer system100 further includes one or more data retrieving device 118 for readingthe data stored on the internal data storage devices 110.

The data retrieving device 118 may represent, for example, a floppy diskdrive, a compact disk drive, a magnetic tape drive, or a modem capableof connection to a remote data storage system (e.g., via the internet)etc. In some embodiments, the internal data storage device 110 is aremovable computer readable medium such as a floppy disk, a compactdisk, a magnetic tape, etc. containing control logic and/or datarecorded thereon. The computer system 100 may advantageously include orbe programmed by appropriate software for reading the control logicand/or the data from the data storage component once inserted in thedata retrieving device.

The computer system 100 includes a display 120 which is used to displayoutput to a computer user. It should also be noted that the computersystem 100 can be linked to other computer systems 125 a-c in a networkor wide area network to provide centralized access to the computersystem 100.

FIG. 11 is a flow diagram illustrating one embodiment of a process 200for comparing a new nucleotide or protein sequence with a database ofsequences in order to determine the homology levels between the newsequence and the sequences in the database. The database of sequencescan be a private database stored within the computer system 100, or apublic database such as GENBANK that is available through the Internet.

The process 200 begins at a start state 201 and then moves to a state202 wherein the new sequence to be compared is stored to a memory in acomputer system 100. As discussed above, the memory could be any type ofmemory, including RAM or an internal storage device.

The process 200 then moves to a state 204 wherein a database ofsequences is opened for analysis and comparison. The process 200 thenmoves to a state 206 wherein the first sequence stored in the databaseis read into a memory on the computer. A comparison is then performed ata state 210 to determine if the first sequence is the same as the secondsequence. It is important to note that this step is not limited toperforming an exact comparison between the new sequence and the firstsequence in the database. Well-known methods are known to those of skillin the art for comparing two nucleotide or protein sequences, even ifthey are not identical. For example, gaps can be introduced into onesequence in order to raise the homology level between the two testedsequences. The parameters that control whether gaps or other featuresare introduced into a sequence during comparison are normally entered bythe user of the computer system.

Once a comparison of the two sequences has been performed at the state210, a determination is made at a decision state 210 whether the twosequences are the same. Of course, the term “same” is not limited tosequences that are absolutely identical. Sequences that are within thehomology parameters entered by the user will be marked as “same” in theprocess 200.

If a determination is made that the two sequences are the same, theprocess 200 moves to a state 214 wherein the name of the sequence fromthe database is displayed to the user. This state notifies the user thatthe sequence with the displayed name fulfills the homology constraintsthat were entered. Once the name of the stored sequence is displayed tothe user, the process 200 moves to a decision state 218 wherein adetermination is made whether more sequences exist in the database. Ifno more sequences exist in the database, then the process 200 terminatesat an end state 220. However, if more sequences do exist in thedatabase, then the process 200 moves to a state 224 wherein a pointer ismoved to the next sequence in the database so that it can be compared tothe new sequence. In this manner, the new sequence is aligned andcompared with every sequence in the database.

It should be noted that if a determination had been made at the decisionstate 212 that the sequences were not homologous, then the process 200would move immediately to the decision state 218 in order to determineif any other sequences were available in the database for comparison.

FIG. 12 is a flow diagram illustrating one embodiment of a process 250in a computer for determining whether two sequences are homologous. Theprocess 250 begins at a start state 252 and then moves to a state 254wherein a first sequence to be compared is stored to a memory. Thesecond sequence to be compared is then stored to a memory at a state256. The process 250 then moves to a state 260 wherein the firstcharacter in the first sequence is read and then to a state 262 whereinthe first character of the second sequence is read. It should beunderstood that if the sequence is a nucleotide sequence, then thecharacter would normally be either A, T, C, G or U. If the sequence is aprotein sequence, then it is preferably in the single letter amino acidcode so that the first and sequence sequences can be easily compared.

A determination is then made at a decision state 264 whether the twocharacters are the same. If they are the same, then the process 250moves to a state 268 wherein the next characters in the first and secondsequences are read. A determination is then made whether the nextcharacters are the same. If they are, then the process 250 continuesthis loop until two characters are not the same. If a determination ismade that the next two characters are not the same, the process 250moves to a decision state 274 to determine whether there are any morecharacters either sequence to read.

If there are not any more characters to read, then the process 250 movesto a state 276 wherein the level of homology between the first andsecond sequences is displayed to the user. The level of homology isdetermined by calculating the proportion of characters between thesequences that were the same out of the total number of sequences in thefirst sequence. Thus, if every character in a first 100 nucleotidesequence aligned with a every character in a second sequence, thehomology level would be 100%.

Homology or identity is often measured using sequence analysis software(e.g., Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705). Such software matches similar sequences byassigning degrees of homology to various deletions, substitutions andother modifications. The terms “homology” and “identity” in the contextof two or more nucleic acids or polypeptide sequences, refer to two ormore sequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same whencompared and aligned for maximum correspondence over a comparison windowor designated region as measured using any number of sequence comparisonalgorithms or by manual alignment and visual inspection.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencefor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443, 1970,by the search for similarity method of person & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444, 1988, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection. Other algorithmsfor determining homology or identity include, for example, in additionto a BLAST program (Basic Local Alignment Search Tool at the NationalCenter for Biological Information), ALIGN, AMAS (Analysis of MultiplyAligned Sequences), AMPS (Protein Multiple Sequence Alignment), ASSET(Aligned Segment Statistical Evaluation Tool), BANDS, BESTSCOR, BIOSCAN(Biological Sequence Comparative Analysis Node), BLIMPS (BLocks IMProvedSearcher), FASTA, Intervals & Points, BMB, CLUSTAL V, CLUSTAL W,CONSENSUS, LCONSENSUS, WCONSENSUS, Smith-Waterman algorithm, DARWIN, LasVegas algorithm, FNAT (Forced Nucleotide Alignment Tool), Framealign,Framesearch, DYNAMIC, FILTER, FSAP (Fristensky Sequence AnalysisPackage), GAP (Global Alignment Program), GENAL, GIBBS, GenQuest, ISSC(Sensitive Sequence Comparison), LALIGN (Local Sequence Alignment), LCP(Local Content Program), MACAW (Multiple Alignment Construction &Analysis Workbench), MAP (Multiple Alignment Program), MBLKP, MBLKN,PIMA (Pattern-Induced Multi-sequence Alignment), SAGA (SequenceAlignment by Genetic Algorithm) and WHAT-IF. Such alignment programs canalso be used to screen genome databases to identify polynucleotidesequences having substantially identical sequences. A number of genomedatabases are available, for example, a substantial portion of the humangenome is available as part of the Human Genome Sequencing Project (J.Roach, http://weber.u.Washington.edu/˜roach/human_genome_progress2.html) (Gibbs, 1995). At least twenty-one other genomes have alreadybeen sequenced, including, for example, M. genitalium (Fraser et al.,1995), M. jannaschii (Bult et al., 1996), H. influenzae (Fleischmann etal., 1995), E. coli (Blattner et al., 1997), and yeast (S. cerevisiae)(Mewes et al., 1997), and D. melanogaster (Adams et al., 2000).Significant progress has also been made in sequencing the genomes ofmodel organism, such as mouse, C. elegans, and Arabadopsis sp. Severaldatabases containing genomic information annotated with some functionalinformation are maintained by different organization, and are accessiblevia the internet, for example, http://wwwtigr.org/tdb;http://www.genetics.wisc.edu; http://genome-www.stanford.edu/˜ball;http://hiv-web.lanl.gov; http://www.ncbi.nlm.nih.gov;http://www.ebi.ac.uk; http://Pasteur.fr/other/biology; and http://www.genome.wi.mit.edu.

One example of a useful algorithm is BLAST and BLAST 2.0 algorithms,which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402,1977, and Altschul et al., J. Mol. Biol. 215:403-410, 1990,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov). This algorithm involves first identifyinghigh scoring sequence pairs (HSPs) by identifying short words of lengthW in the query sequence, which either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighborhood wordscore threshold (Altschul et al., supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are extended in both directions alongeach sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a word length (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a word lengthof 3, and expectations (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Natl. Acad. Sci. USA 90:5873, 1993). One measure of similarity providedby BLAST algorithm is the smallest sum probability (P(N)), whichprovides an indication of the probability by which a match between twonucleotide or amino acid sequences would occur by chance. For example, anucleic acid is considered similar to a references sequence if thesmallest sum probability in a comparison of the test nucleic acid to thereference nucleic acid is less than about 0.2, more preferably less thanabout 0.01, and most preferably less than about 0.001.

In one embodiment, protein and nucleic acid sequence homologies areevaluated using the Basic Local Alignment Search Tool (“BLAST”) Inparticular, five specific BLAST programs are used to perform thefollowing task:

-   -   (1) BLASTP and BLAST3 compare an amino acid query sequence        against a protein sequence database;    -   (2) BLASTN compares a nucleotide query sequence against a        nucleotide sequence database;    -   (3) BLASTX compares the six-frame conceptual translation        products of a query nucleotide sequence (both strands) against a        protein sequence database;    -   (4) TBLASTN compares a query protein sequence against a        nucleotide sequence database translated in all six reading        frames (both strands); and    -   (5) TBLASTX compares the six-frame translations of a nucleotide        query sequence against the six-frame translations of a        nucleotide sequence database.

The BLAST programs identify homologous sequences by identifying similarsegments, which are referred to herein as “high-scoring segment pairs,”between a query amino or nucleic acid sequence and a test sequence whichis preferably obtained from a protein or nucleic acid sequence database.High-scoring segment pairs are preferably identified (i.e., aligned) bymeans of a scoring matrix, many of which are known in the art.Preferably, the scoring matrix used is the BLOSUM62 matrix (Gonnet etal., Science 256:1443-1445, 1992; Henikoff and Henikoff, Proteins17:49-61, 1993). Less preferably, the PAM or PAM250 matrices may also beused (see, e.g., Schwartz and Dayhoff, eds., 1978, Matrices forDetecting Distance Relationships: Atlas of Protein Sequence andStructure, Washington: National Biomedical Research Foundation). BLASTprograms are accessible through the U.S. National Library of Medicine,e.g., at www.ncbi.nlm.nih.gov.

The parameters used with the above algorithms may be adapted dependingon the sequence length and degree of homology studied. In someembodiments, the parameters may be the default parameters used by thealgorithms in the absence of instructions from the user.

FIG. 13 is a flow diagram illustrating one embodiment of an identifierprocess 300 for detecting the presence of a feature in a sequence. Theprocess 300 begins at a start state 302 and then moves to a state 304wherein a first sequence that is to be checked for features is stored toa memory 115 in the computer system 100. The process 300 then moves to astate 306 wherein a database of sequence features is opened. Such adatabase would include a list of each feature's attributes along withthe name of the feature. For example, a feature name could be“Initiation Codon” and the attribute would be “ATG”. Another examplewould be the feature name “TAATAA Box” and the feature attribute wouldbe “TAATAA”. An example of such a database is produced by the Universityof Wisconsin Genetics Computer Group (www.gcg.com). Alternatively, thefeatures may be structural polypeptide motifs such as alpha helices,beta sheets, or functional polypeptide motifs such as enzymatic activesites, helix-turn-helix motifs or other motifs known to those skilled inthe art.

The following examples are intended to illustrate, but not limit, theinvention.

EXAMPLE 1 Identification of Proteins Interacting With GlutamateTransporter Proteins

Yeast Two-Hybrid with EAAT4 Yeast two-hybrid screens were performedusing the HF7c′ yeast strain harboring the reporter genes HIS 3 andβ-galactosidase (β-gal) under the control of GAL4 activation. The final77 amino acids of EAAT4 (carboxy-intracellular domain SEQ ID NO: 12)were subcloned in-frame into pGBT9 (GAL4 binding domain vector,CLONTECH) and used to screen a rat brain cDNA library constructed inpGAD10 (GAL4 activation domain vector, CLONTECH). The plasmids weretransformed into HF7c′ yeast cells and positive clones selected ontriple-minus plates (Leu-, Trp-, His-) and assayed for β-galactosidaseactivity. Positive clones were co-transformed with either the baitvector or the original pGAD10 vector into yeast cells to confirm theinteraction. For a subsequent EAAT4 C-terminal domain analysis,different regions of the final 77 amino acids of EAAT4 were subclonedin-frame into the pGBT9 vector.

Yeast Two-Hybrid Screen with EAAC1 The MATCHMAKER Two-Hybrid System(Clontech) was used for screening. Using the carboxy-terminalintracellular domain of EAAC1 (the carboxy-87 amino acids, cDNA position1458-1719; SEQ ID NO: 13) as bait in a yeast two-hybrid screen of anadult rat brain cDNA library, 78 clones displaying β-galactosidaseactivity were identified. Plasmid DNAs were isolated from positiveclones and re-co-transformed with bait cDNA back into yeast to reconfirmthe interaction. Restriction and sequencing analyses revealed that tenof these clones with the strongest β-galactosidase activity wereidentical.

EXAMPLE 2 Isolation and Primary Structure of Glutamate TransporterAssociated Proteins

Cloning of full-length GTRAP4-41 and GTRAP4-48 cDNAs. Marathon cDNAamplification (CLONTECH) was used to perform both 5′- and 3′-RACE oncDNA synthesized from rat brain poly(A)⁺ RNA. The double-stranded cDNAwas ligated to the Marathon cDNA Adaptor which contains an adaptorprimer (AP1) binding site. The 1.1 kb GTRAP4-41and 1.4 kb GTRAP4-48 cDNAfragments identified using the yeast two-hybrid system were used todesign gene-specific primers (GSPs) which could be used in 5′- and3′-RACE PCR reactions along with the AP1 primer. The RACE productsobtained were sequenced and new GSPs designed, generating a series ofoverlapping RACE products, which were joined together by PCR.Overlapping RACE products were put through ten cycles of denaturation,annealing and extension in the absence of primers. Nested primers wereadded and the PCR continued for a further 20 cycles to amplify theoverlapped template.

GTRAP4-41 AND 4-48 Two independent cDNA clones were isolated and theproteins they encode were named GTRAP4-41 and GTRAP4-48 (for glutamatetransporter 4 associated protein). Isolation of the full-length cDNAs bya series of 5′ and 3′ RACE PCR reactions demonstrated that the largestopen reading frame (ORF) for GTRAP4-41 is 7,164 base pairs (SEQ ID NO:1), which encodes a 2,388 amino acid protein (SEQ ID NO:2) with apredicted relative molecular mass (M_(r)) of 270,958 Da (accessionAF225960). A BLAST search of the GenBank database shows that GTRAP4-41possesses 87% identity with β-spectrin III (accession AB008567).GTRAP4-41 possesses seventeen 16 amino acid spectrin repeats, twoα-actinin domains and a pleckstrin homology domain (FIG. 1A).

The largest ORF identified for GTRAP4-48 (accession AF225961) is 4,581base pairs (SEQ ID NO:3), which encodes a 1,527 amino acid protein (SEQID NO:4) with a predicted M_(r) of 168,698 Da. A BLAST search of theGenBank database shows that GTRAP4-48 is unique, but it possessessignificant homology to the KIAAO380 cDNA-encoded protein (90% identity)and the recently described RhoGEF, p115. GTRAP4-48 posses multiplepotential interaction and regulatory domains (FIG. 1B). GTRAP4-48 has aPDZ domain, a regulatory G-protein domain, a pleckstrin homology region(PH) and two proline-rich sequences (PRO). These regions have all beenimplicated in protein-protein interactions by interacting with the Ctermini of proteins and are thought to be important in the subcellulartargeting of the interacting proteins (Katan et al., FEBS Lett. (1999)452:36-40; LeVine Mol.Neurobiol. (1999); 19:111-149). The function of PHdomain is not clearly clarified, several putative functions have beensuggested; (1) binding to the β/γ subunit of heterotrimeric G proteins,(2) binding to lipids, (3) binding to phosphorylated Ser/Thr residues,(4) attachment to membrane by an unknown mechanism. The protein multiplePKC phosphorylation sites and one tyrosine kinase phosphorylation site.It contains 3 helix- loop-helix signatures. Finally, the proteincontains a separated tandem periodic repeat of SQPEGS of underminedsignificance.

GTRAP3-18. Following isolation, one clone, E18, was completely sequencedand the protein encoded is named GTRAP3-18 (glutamate transporter EAAC1associated protein). Clone E18 is a full-length cDNA containinginitiation methionine and polyA tail (SEQ ID NO:5). GTRAP3-18 encodes aprotein of 188 amino acids (SEQ ID NO:6) with a calculated molecularmass of 22.5 kDa. Protein analysis indicates that it is a veryhydrophobic protein with four possible transmembrane domains. Both thecarboxy-terminal and amino-terminal domains contain protein kinase Cmotifs and may be intracellular. JWA protein (Genbank NP006398), a novelhuman differentially displayed vitamin A responsive gene, is 95%identical to GTRAP3-18, suggesting that GTRAP3-18 is a rat JWA proteinhomologue.

EXAMPLE 3 Antibodies

Generation of Polyclonal GTRAP4-41 and GTRAP4-48 Antibodies. Affinitypurified polyclonal antisera to EAAT4, GTRAP4-41 and GTRAP4-48 wereproduced using methods identical to previous studies (Rothstein et al.,Neuron (1994) 13:713-725. Synthetic peptides corresponding to epitopesof EAAT4 (carboxy-terminal; EKGASRGRGGNESA; SEQ ID NO:14 andamino-terminal; KNSLFLRESGAGGGCL; SEQ ID NO:15), rat GTRAP4-41(KRGPAPSPMPQSRSSE; SEQ ID NO:16) and rat GTRAP4-48 (KTPERTSPSHHRQPSD;SEQ ID NO:17) were synthesized. Monospecific antibodies to GTRAP4-41 and4-48 were produced.

The affinity-purified GTRAP4-41 antibodies recognize a 270 KDa proteinin HEK 293T cells transfected with the full-length GTRAP4-41 cDNA andthe affinity-purified GTRAP4-48 antibodies recognized a 170 KDa proteinin HEK 293T cells transfected with the full-length GTRAP4-48 cDNA

Generation of Polyclonal GTRAP3-18 Antibodies. Affinity purifiedpolyclonal antisera to GTRAP3-18 was produced as described in Rothsteinet al. (1994) using the amino terminal region epitope, (KFFPGSDRFARPDFRSEQ ID NO: 18).

EXAMPLE 4 Expression of Glutamate Transporter Associated Proteins

Fusion proteins and in vitro binding. Full-length EAAT4 was subclonedinto the EcoR I site of the GST-fusion vector pGEX-6P-1(Pharmacia).Synthesis of recombinant proteins in BL21 cells (Novagen) was induced by0.1 mM isopropyl β-D-thiogalactoside for 2 hrs at 30° C. and purifiedaccording to the protocol provided by the manufacturer (Pharmacia). HEK293T cells were transfected with myc-tagged GTRAP4-41 or GTRAP4-48 andharvested in ice-cold immunoprecipitation (IP) buffer (phosphatebuffered saline (pH 7.1), 5 mM EDTA, 1 mM sodium orthovanadate, 0.1 mMphenylmethylsulphonyl fluoride (PMSF), 0.3 μM aprotinin and 1% TritonX-100). The cellular lysate was incubated with GST or GST-EAAT4immobilized on glutathione-Sepharose-4B, and washed to removenon-specifically bound proteins. Specifically bound proteins were elutedwith 2×SDS loading buffer and analyzed by immunoblotting using ananti-c-myc antibody.

The Glutathione S-transferase (GST) Gene Fusion System (Pharmacia) wasused to construct and generate GST-EAAC1 and GST-GTRAP3-18 fusionproteins using pGEX-6P-1 vector as described herein.

GTRAP4-41 and 4-48 Expression Constructs. For transient expression inHEK 293T cells full-length EAAT4 cDNA was subcloned into the EcoR I/BamHI site of the mammalian expression vector pRK5 (Genentech). Forco-immunoprecipitation full-length GTRAP4-41 and GTRAP4-48 cDNAs weresubcloned into the Not I site of a myc-tagged pRK5 vector.

Cell culture and cell transfection. HEK 293T cells were obtained fromthe American Type Culture Collection (Rockville, Md.) and maintained inMEM medium supplemented with 10% fetal bovine serum and L-glutamine. Fortransient transfections cells, were pre-washed with phosphate bufferedsaline (PBS) and incubated for 10 min at 4° C. with 40 mg of eachplasmid DNA and 20 mg of salmon sperm DNA. Cells were transfected byelectroporation at 300 V and 500 μF with a gene pulser (Bio-Rad) andgrown for 48-72 h in either 10 cm culture dishes or plated ontopoly-D-lysine coated coverslips in 6-well plates for co-localizationstudies.

Subcloning, stable transfection and maintenance of cell lines The EAAT4cDNA was subcloned into pcDNA3.1/Hygro(+) (Invitrogen) using the EcoR Irestriction site. The plasmid was linearized with Ssp I, ethanolprecipitated and transfected into HEK 293T cells using the calciumphosphate-DNA precipitation method. 50 mg of DNA per 10 cm dish wasused. Cells were incubated with the precipitate in 5% CO₂ at 37° C. for6 hours, the medium containing the precipitate was removed and the cellswere washed twice with PBS before adding fresh MEM medium. 48 h aftertransfection, the cells were split to 50% confluency and hygromycin(Invitrogen) was added at a concentration of 50 mg/ml. Cell culturemedium containing hygromycin was changed every 3 to 4 days. Afterapproximately 2- to 3-weeks of selection, a serial dilution was carriedout and cells were plated out, without selection, in a 96-well plate toobtain one cell per well. Several colonies were picked, expanded inselective medium and checked for expression by western blotting.Similarly, the GTRAP4-41 CDNA was cloned into pcDNA3 using the Not Irestriction site and linearized with Ssp I. Selection was with G418(Mediatech) at a concentration of 5 mg/ml. The GTRAP4-48 cDNA was clonedinto the inducible expression vector pIND (Invitrogen) using the EcoR Irestriction site and linearized with Sca I. Selection was with G418 andexpression of GTRAP4-48 was induced with 5 mM Ponasterone A(Ecdysone-Inducible Mammalian Expression System, Invitrogen).

Co-immunoprecipitation in heterologous cells. Full-length GTRAP4-41 andGTRAP4-48 cDNAs were subcloned into the Not I site of a myc-tagged pRK5vector and used to transfect the HEK-rEAAT4 cell line by electroporationat 300 V and 500 μF with a gene-pulser (Bio-Rad). After transfection(48-72 h), cells were solubilized with 1 ml of ice-cold IP buffer for 2h at 4° C. with rotation and centrifuged to remove cellular debris. 5 μgof rabbit anti-NEAAT4 antibody was added to 0.5 ml of supernatant andincubated overnight at 4° C. 150 μl protein A-Sepharose (Pharmacia) wasthen added for 2 h at 4° C., washed once with IP buffer and three timeswith IP minus Triton X-100. Bound protein was eluted by boiling in 3×SDSloading buffer, and analyzed by immunoblotting using the anti-c-mycantibody.

GTRAP3-18 Expression. For transient expression in HEK 293T cells,full-length EAAC1 cDNA was subcloned into the EcoR I/BamH I site of themammalian expression vector pRK5 (Genentech). For co-immunoprecipitationfull-length GTRAP3-18 cDNA were subcloned into the Not I site of amyc-tagged pRK5 vector.

Cell culture and cell transfection of GTRAP3-18 For transienttransfections cells were pre-washed with PBS, incubated for with 40 mgof each plasmid DNA /20 mg of salmon sperm DNA and electroporated asdescribed herein. In some cases, C6 glioma cells, known to naturallyexpress high levels of EAAC1, were transfected with GTRAP3-18.

EXAMPLE 5

To determine the biochemical interaction between Glutamate TransporterAssociated Proteins and glutamate transporter proteins, binding andimmunoprecipitation assasys in vivo and in vitro were performed.

GTRAP4-41 and 4-48 Immunoprecipitation with Fusion Proteins. Full-lengthEAAT4 was subcloned into the EcoR I site of the GST-fusion vectorpGEX-6P-1 (Pharmacia). Synthesis of recombinant proteins in BL21 cells(Novagen) was induced by 0.1 mM isopropyl b-D-thiogalactoside for 2 hrsat 30° C. and prepared as a crude bacterial lysate by mild sonication inice-cold 1 C PBS and solubilization in 1% Triton X-100. Cell debris wasremoved by centrifugation at 7,000 g and the cleared bacterial lysateapplied to glutathione-Sepharose-4B (Pharmacia). HEK 293T cells weretransfected with myc-tagged GTRAP4-41 or GTRAP4-48 as described hereinand harvested in ice-cold immunoprecipitation (IP) buffer (phosphatebuffered saline (pH 7.1), 5 mM EDTA, 1 mM sodium orthovanadate, 0.1 mMphenylmethylsulphonyl fluoride (PMSF), 0.3 mM aprotinin and 1% TritonX-100) with 1% Triton X-100. The cellular lysate was incubated with GSTor GST-EAAT4 immobilized on glutathione-Sepharose-4B, and washed toremove non-specifically bound proteins. Specifically bound proteins wereeluted with 3×SDS loading buffer and analyzed by immunoblotting using ananti-c-myc antibody. Bands were visualized by HRP-conjugated secondaryantibodies and ECL chemiluminescence (Amersham).

GTRAP4-41 and GTRAP4-48 bind to GST-EAAT4 fusion protein, but do notbind to GST.

Co-immunoprecipitation in heterologous cells Transiently transfectedcells (as described herein) were solubilized with 1 ml of ice-cold IPbuffer for 2 h at 4° C. with rotation and centrifuged to remove cellulardebris. 1.2 mg of mouse anti-c-myc antibody was added to 0.5 ml ofsupernatant and incubated overnight at 4° C. 150 ml protein A-Sepharose(Pharmacia) was then added for 2 h at 4° C., washed once with IP bufferand three times with IP minus Triton X-100. Bound EAAT4 was eluted byboiling in 3×SDS loading buffer, and analyzed by immunoblotting usingthe anti-carboxy-terminal EAAT4 antibody.

GTRAP4-41, GTRAP4-48 and KIAA0380 (a close homolog of GTRAP4-48) arecoimmunoprecipitated with EAAT4 protein using the amino-terminalanti-EAAT4 antibody.

Immunoprecipitation from cerebellum lysate. Sprague-Dawley (SD) ratcerebellum was dissected, washed with 50 mM Tris-HCl (pH 7.5), 2 mM EDTAand 0.5 mM DTT, and homogenized on ice in buffer containing 20 mMTris-HCl (pH 7.5), 10% sucrose, 1 mM EDTA, 0.1 mM PMSF, 0.3 mMaprotinin, 1 mM benamidine, 10 mg/ml leupeptine and 10 mg/ml pepstatine.Protein concentration was measured and adjusted to 2-3 mg/ml, and thehomogenate was mixed in a 1:1 ratio with the solubilization buffer(homogenization buffer plus 2% Triton X-100). After 2 h, the lysate wasspun at 10,000 g for 10 min. For each immunoprecipitation, 500 mg of theTriton-lysate was incubated overnight at 4° C. with 5 μg of theanti-amino-terminal EAAT4 antibody. Immune complexes were precipitatedwith protein A Sepharose (Pharmacia), washed three times with 10 mMTris-HCl (pH 7.5) and 5 mM EDTA, eluted with 3×SDS loading buffer, andprocessed for western blot analysis. The filters were probed withaffinity purified rabbit antibodies against GTRAP4-41 and GTRAP4-48.

The biochemical interaction between GTRAP4-41 or GTRAP4-48 and EAAT4 wasconfirmed using an in vitro binding assay. Full-length myc-taggedGTRAP4-41 and GTRAP4-48 were expressed in HEK 293T cells. Thesolubilized cell extracts were then mixed with bead-linked GST-EAAT4 orGST alone and the bound proteins were eluted. GTRAP4-41 and GTRAP4-48were specifically retained by the GST-EAAT4 fusion protein, but not byGST alone.

To further assess the interaction between GTRAP4-41 or GTRAP4-48 andEAAT4 in a cellular context, immunoprecipitation studies in transfectedheterologous cells were performed. A stable rat EAAT4 expressing cellline was generated in HEK 293T cells (HEK-rEAT4) and transfected withcDNAs encoding myc-tagged GTRAP4-41 and GTRAP4-48. Antibodies directedto the amino-terminus of EAAT4 were used to immunoprecipitate theantigen and any associated protein.

Western blot analysis using an anti-c-myc antibody demonstrates thatGTRAP4-41 and GTRAP4-48 coimmunoprecipitate with EAAT4. Nocoimmunoprecipitation is observed when the precipitating antibody isomitted. Similarly, when the anti-c-myc antibody is used, EAAT4 isco-immunoprecipitated with GTRAP4-41 and GTRAP4-48.

The GTRAP4-41 and GTRAP4-48 interaction with EAAT4 was then studied invivo using solubilized brain preparations. GTRAP4-41 and GTRAP4-48 areco-immunoprecipitated with EAAT4 from brain by antibodies directed atthe amino-terminus of EAAT4 but not by antibodies directed at thecarboxy-terminus. However, since the site of interaction is within thecarboxy-terminus of EAAT4, it is likely that the carboxy-terminalantibodies disrupt the protein-protein interaction. Furthermore,GTRAP4-41 and GTRAP4-48 appear to specifically interact with EAAT4, asGTRAP4-41 and GTRAP4-48 do not co-immunoprecipitate from brain withantibodies directed at the other glutamate transporters, e.g., GLT-1,GLAST and EAAC1. GTRAP4-48 is also not co-immunoprecipitated from brainwith antibodies directed at GTRAP4-41. Similarly, GTRAP4-41 is notco-immunoprecipitated with antibodies directed at GTRAP4-48 from HEK293T cells that were transfected with full-length myc-tagged GTRAP4-41and GTRAP4-48, indicating that there is no direct interaction betweenGTRAP4-41 and GTRAP4-48.

GTRAP3-18 Immunoprecipitation. Coronal sections of rat brain were slicedat 1-2 mm intervals from the cerebellum to the olfactory bulbs. Thecortex region was excised from the brain and placed in cold buffer A (50mM Tris pH 7.5, 2 mM EDTA, 150 mM NaCl, 0.5 mM DTT). The tissue waswashed three times in buffer A and the tissue was weighted. The tissuewas then homogenized using a blender in 2.5 vol of buffer B (50 mM TrispH 7.5, 10% glycerol, 5 mM Mg acetate, 0.2 mM EDTA, 0.5 mM DTT, 1 mMPMSF). The particulate material was removed by centrifugation at15,000×g for 30 min at 4° C. The supernatant fraction was incubated withProtein A Sepharose beads and primary antibodies as described herein.

The interaction of GTRAP3-18 with EAAC1 was examined using in vitro andin vivo methods. For in vitro cell-free binding, EAAC1 was expressed asa fusion protein with glutathione S-transferase (GST), and GTRAP3-18 wasproduced and labeled with [³⁵S]-methionine by in vitro transcription andtranslation. Purified GST or GST-EAAC1 fusion proteins immobilized onglutathione-Sepharose were incubated with [³⁵S]-labeled GTRAP3-18protein. GTRAP3-18 specifically binds to immobilized GST-EAAC1 but notto GST alone, indicating an in vitro interaction.

Immunoprecipitation experiments were performed to test if EAAC1 andGTRAP3-18 interact in vivo. This was first examined in transfectedHEK293 cells using amino-terminus c-myc tagged GTRAP3-18. EAAC1 isco-immunoprecipitated with c-myc-GTRAP3-18 in the cell extract preparedfrom co-expression cells but not from EAAC1 or c-myc-GTRAP3-18 singleexpression cells. Theses studies with single expression cells show thatbinding is specific since they rule out the possibility that the resultsare due to artifact from immunobead nonspecific binding or antibodycross-reaction, respectively. A truncated EAAC1 lacking the interactingcarboxy-terminal domain (described herein) is not co-immunoprecipitatedwith c-myc-GTRAP3-18, further demonstrating the interaction of EAAC1 andGTRAP3-18. This interaction was specific, since EAAT4, another neuronalglutamate transporter subtype, is not immunoprecipitated withc-myc-GTRAP3-18. Identical results are obtained using COS-7 and C6glioma cell lines.

To study the protein interaction in vivo, anti-EAAC1 or GTRAP3-18polyclonal antibodies were used to immunoprecipitate EAAC1 or GTRAP3-18from rat brain extract. Western blotting demonstrates that EAAC1 isspecifically co-immunoprecipitated with GTRAP3-18, but not with GLAST,GLT-1 or EAAT4. Similarly, GTRAP3-18 was co-immunoprecipitated withEAAC1. These studies suggest that EAAC1 and GTRAP3-18 interact in vivo.

EXAMPLE 6

Identification of the EAAT4- GTRAP4-48 interaction site To evaluate thegeneral/region and or amino acid motif required for the association ofGTRAP 4-41 and GTRAP4-48 with EAAT4, a series of two-hybrid screen usingdifferent EAAT4, carboxy-terminal truncations and GTRAP proteins as baitwas performed. A series of successively larger carboxy terminaldeletions of the 77-amino acid carboxy-terminal EAAT4 bait was used toidentify regions necessary for ginding of GTRAP4-41 and GTRAP4-48 toEAAT4. Residues 555-561 (QEAELTLP; SEQ ID NO:9) and 527-534 (GRGGNESVM;SEQ ID NO:10) are required for GTRAP4-41 and GTRAP4-48 binding,respectively (FIG. 2).

EXAMPLE 7 Expression and Localization of GTRAP

Expression of GTRAP4-41 and 4-48 protein in brain. GTRAP4-41 andGTRAP4-48 are expressed exclusively in the brain. The highest level ofexpression of both proteins is in the cerebellum, and somewhat lowerlevels of expression in the cortex. The apparent molecular weight forGTRAP4-41 is greater than 201 kD; the apparent molecular weight forGTRAP4-48 is less that 200 kD. The native proteins migrate in PAGEidentical to proteins expressed in transfected HEK cells

Expression of GTRAP3-18 mRNA in brain Northern analyses of GTRAP3-18mRNA were performed in brain as well as various body organ tissues.Total RNA was isolated from various rat tissues using a Stratagene RNAisolation kit, separated on 1% agarose gel with 6.7% formaldehyde andblotted onto a nylon membrane (Gene-screen Plus; NEN) by capillarytransfer. The blot was hybridized to the full length cDNA probe labeledwith ³²p by random priming, washed for 2×10 min in 2×SSC, 0.1% SDS at42° C., 1×20 min in 0.1×SSC, 0.1% SDS at 65° C. and autoradiographedovernight.

GTRAP3-18 mRNA is widely distributed; GTRAP3-18 mRNA is found in thebrain, kidney, heart, muscle, liver and cortex. This pattern isconsistent with the distribution of EAAC1 in peripheral tissues (Furutaet al., J Neuroscience (1997) 17:8363-8375; Shayakul et al.,Amer.J.Physiol.Renal Physiol. (1997) 42:F1023-F1029). Similarly,GTRAP3-18 protein is expressed in many neural and non-neural tissues,based on immunocytochemical studies using a polyclonal oligopeptideantibody to the amino-terminus of GTRAP3. GTRAP3-18 protein appears toaggregate as multimers. The dimeric form of GTRAP3-18 is the predominantspecies in tissue homogenates. The dimeric form is also observed whenpurified GTRAP3-18 protein is detected using the amino-terminalGTRAP3-18 antibody, and when c-myc-GTRAP3-18 protein is detected usinganti-c-myc antibodies. Immunohistological analysis of rat brain revealsthat GTRAP3-18 protein is expressed widely and is primarily localized toneurons such as cerebellar Purkinje cells which is identical to thedistribution of EAAC1. In transfected HEK293 cells, EAAC1 proteinappears to be aggregated at the cell membrane, while GTRAP3-18 proteinis typically localized to the cell membrane and cytosol, andco-associated with EAAC1 protein at the cell membrane.

Co-localization of GTRAP4-48 or GTRAP4-41 with EATT4 HEK cells,transientlytransfected with EAAT4 (20 μg) and/or GTRAP4-41 (20 μg)and/or GTRAP4-48 (20 μg), were fixed with paraformaldehyde (4%) inphosphate buffer (0.1 M, pH7.4) for 20 min. The cells were thenpermeabilized with 0.1 % Triton X-100, stained with the primaryantibodies EAAT4 (1 mg/ml) and c-myc (5 mg/ml) for 1 h, rinsed andincubated with Texas-red and FITC-conjugated secondary antibodies atdilutions of 1:200. Immunofluorescence was viewed with a confocalmicroscope. Confocal microscopy of transfected cells and of brainsections was performed with a Zeiss LSM 510 laser scanning microscopeusing fluorescein (Vector, #FI1000) and Texas red (Vector, TI2000)flurochromes.

Both GTRAP4-41 and 4-48 co-localize to membranes domains with EAAT4.GTRAP4-48 expression is associated with a re-distribution of the proteinon the membrane in a punctate-like organization.

Immunohistochemistry The cellular localization of GTRAP4-41 and 4-48 wasstudied in rat brain tissue. Rat brain sections were stained, aspreviously described Furuta, A. et al., Neuroscience 81, 1031-1042(1997)) using the following antibodies: carboxy-terminal anti-EAAT4 (1.5μg/ml), anti-GTRAP4-41 (127 ng/ml) or anti-GTRAP4-48 (132 ng/ml)antibodies. Texas-red and FITC-conjugated secondary antibodies were usedat dilutions of 1:200.

Both GTRAP4-41 and GTRAP4-48 are highly localized to rat cerebellarcortex, with prominent immunolocalization to Purkinje cell somas anddendrites.

GTRAP4-41 and GTRAP4-48 are selectively localized to brain. In rat,GTRAP4-41 and GTRAP4-48 are predominately expressed in the cerebellum,especially the cerebellar cortex with prominent immunolocalizationobserved in Purkinje cell somas and dendrites, with low levelimmunoreactivity in striatum, hippocampus and thalamus. Previous studieshave shown that EAAT4 is selectively localized to cerebellar Purkinjecells, although low level expression is observed in cerebral cortex,hippocampus and striatum (Furuta et al., Neuroscience 81, 1031-1042(1997)) Thus, GTRAP4-41, GTRAP4-48 and EAAT4 are co-localized in braintissue.

GTRAP3-18 mRNA is widely expressed in brain regions and body organs,consistent with the distribution of EAAC1 (Hediger et al., Am. J Physiol277, F477-F480 (1999); Hediger Am. J Physiol 277, F487-F492 (1999)).Similarly, GTRAP3-18 protein is expressed in many neural and non-neuraltissues when protein localization is examined using a polyclonaloligopeptide antibody to the amino-terminus of GTRAP3. GTRAP3-18 proteinappears aggregated as multimers. The dimeric form of GTRAP3-18 is thepredominant species in tissue homogenates and the dimeric form is alsoobserved when purified GTRAP3-18 protein is detected using theamino-terminal GTRAP3-18 antibody, as well as when c-myc-GTRAP3-18protein is detected using anti-c-myc antibodies. Immunohistologicalanalysis of rat brain reveals that GTRAP3-18 protein is expressed widelyand primarily localized to neurons such as cerebellar Purkinje cells,identical to the expression pattern observed for EAAC1 (He et al., JComp Neurol 418, 255-269 (2000); Rothstein et al. Neuron 13, 713-725(1994)).

Localization in heterologous cells In transfected HEK293 cells, EAAC1protein appears aggregated at the cell membrane while GTRAP3-18 proteinis typically localized to the cell membrane and cytosol, andco-associated with EAAC1 protein at the cell membrane.

EXAMPLE 8 GTRAPs Modulate Glutamate Transport

To determine function relationships between GTRAPs and glutamatetransporter proteins, sodium-dependent, glutamate transport activity wasmeasured in HEK-rEAAT4 cells transfected with one or more interactingproteins.

To determine the effects of GTRAPs on glutamate transport function,Na+-dependent glutamate transport activity was measured in cells stablytransfected with EAAT4, EAAC1, or another glutamate transporter proteinand one or more interacter proteins. Stably transfected cells were grownin a monolayer on 6-well plates in MEM supplemented with 10% fetalbovine serum and L-glutamine. Assays were conducted when cells reached˜80% confluency. The wells were pre-rinsed twice with 2 ml of ice-coldtissue buffer (50 mM Tris, 320mM sucrose, pH 7.4). The cells were thenincubated for 4 min at 37° C. with 1 ml of either sodium- (120 mM NaCl,25 mM NaHCO₃, 5 mM KCl, 2 mM CaCl₂, 1 mM KH₂PO₄, 1 mM MgSO₄, 10% glucoseand 10 mM glutamate) or choline- (120 mM choline Cl, 25 mM Tris-HCl pH7.4, 5 mM KCl, 2mM CaCl₂, 1 mM KH₂PO₄, 1 mM MgSO₄, 10% glucose and 10 mMglutamate) containing buffer labeled with L-[³H]Glu. Uptake was stoppedby rinsing three times with 2 ml of ice-cold wash buffer (50 mM Tris pH7.5 and 160 mM NaCl). Cells were solubilized in 1 ml of 0.1 N NaOH, and500 ml of lysate was analyzed for radioactivity in a scintillationcounter. Na⁺-dependent uptake was defined as the difference inradioactivity accumulated in Na⁺-containing buffer and incholine-containing buffer. Protein content was measured and glutamateuptake calculated as nmole glutamate per mg of protein. In some cases,homogenates were examined for EAAT4, GTRAP4-41 and GTRAP 4-48 protein.

In an alternative protocol, HEK-rEAAT4 cells transfected with GTRAP4-41and GTRAP4-48 were grown in a monolayer on 6-well plates in MEMsupplemented with 10% fetal bovine serum and L-glutamine. Assays wereconducted about 72 h after transfection using the method of Davis (Daviset al., J. Neurosci. 18, 2475-2485 (1998)). GTRAP4-41 and GTRAP4-48 weresubcloned into the Eco RI site of HSV PrPUC amplicon parent vector(pHSVPrPUC) (Geller, A. I., et al., Proc Natl Acad Sci U.S.A 87,8950-8954 (1990)). 3.6 μg of amplicon vector DNA and 25 μg pBAC-V2 DNAwere used to transfect 2-10⁷ baby hamster kidney cells according topreviously published methods (Stavropoulos and Strathdee, J. Virol. 72,7137-7143 (1998)). Virus was harvested about 72 hrs after transfectionand titered as previously described (Bowers et al. Mol. Ther. 1, 294-299(2000))²⁴. 2×10⁵ expression particles were injected intra-cistemallyinto male Sprague-Dawley rats (250 g) obtained from Zivic Miller. About48 h after injection, the rats were sacrificed and synaptosomalpreparations of the cerebelli were prepared using a polytron. Glutamatetransport was measured by methods described herein.

GTRAP4-41 and GTRAP4-48 produce a two- to four-fold increase inglutamate transport, respectively. The co-expression of GTRAP4-41 andGTRAP4-48 results in a further increase in glutamate uptake. Kineticanalysis indicates that GTRAP4-41 and GTRAP4-48 produced an increase inthe Vmax of glutamate transport activity (FIG. 3). There is also a smallincrease in the K_(m) values when GTRAP4-41 and GTRAP4-48 areco-expressed, but these are not statistically significant, suggestingthat the interacting proteins do not alter the affinity of thetransporter for glutamate. GTRAP4-41 and GTRAP4-48 may therefore enhanceglutamate transport either via an increase in the catalytic rate of thetransporter or via an increase in cell surface availability. Results arepresented in FIG. 3. GTRAP41 and GTRAP48 expression in HEK-rEAAT4 cellsincrease glutamate uptake significantly over vector alone (VA)transfected cells. Data in FIG. 3A are the mean±SEM of at least fourindependent observations and were compared by students t test, (**p<0.005). Concentration dependence of Na⁺-dependent L-[³H]-glutamateuptake was assayed in the presence of increasing concentrations ofglutamate. In FIG. 3B, the values are expressed as the mean±SEM of arepresentative experiment carried out in triplicate. Kinetic data showsthat GTRAP41 ( ) increases the V_(max) from 222 to 605 pmol/mg/min andincreases the K_(m) slightly from 7 to 11 μM, compared to EAAT4 alone(>). GTRAP48 increases the V_(max) from 208 to 512 pmol/mg/min (•) andincreased the K_(m) from 10 to 13 μM.

To test if GTRAP3-18 modulates EAAC1 function, sodium-dependent[³H]-glutamate transport was studied in HEK293 cells co-expressing bothproteins, 72 hrs after transfection (Rothstein et al. Neuron 16, 675-686(1996); Lin et al., Neuron 20, 589-602 (1998)). Total glutamatetransport progressively decreases with increasing GTRAP3-18 proteinexpression (FIG. 4). GTRAP3-18 negatively modulates EAAC1-mediatedglutamate transport. Glutamate transport was studied in HEK293 cellstransfected with plasmids indicated in FIG. 4. GTRAP3-18 inhibitedEAAC1-mediated transport, but had no effect on EAAT4 (n=6). Theco-expression of GTRAP-3-18 has no effect on total EAAC1 proteinexpression. Analysis of HEK293 cells by confocal microscopy and surfacebiotinylation reveal no alteration in the membranous localization ofEAAC1. Superoxide dismutase (SOD1) was used as a control.Eadie-Scatchard plot of glutamate transport in transfected HEK293 cellsreveals a 4-10 fold decrease in affinity (n=4). This effect is specificfor EAAC1; co-expression of GTRAP3-18 with EAAT4 has no effect ontransport activity. The inhibition of transport is not due to a decreaseof EAAC1 protein level by the co-expression of GTRAP3-18, as quantitatedby Western blotting. Similarly, the loss of EAAC1 activity is not due toaltered protein trafficking; even at high levels of GTRAP3-18expression, when little EAAC1-mediated transport is observed, EAAC1surface expression is unaltered as determined by surface biotinylationand confocal microscopy.

To evaluate the biochemical nature of altered transport, kineticanalyses were performed with HEK293 cells co-expressing EAAC1 andGTRAP3-18. EAAC1 and GTRAP3-18 co-expressing cells show a decrease inaffinity (K_(m)=40 μM, Vmax=0.99 nmol/min/mg protein; n=4, P<0.01)without a shift in the V_(max) when compared to cells only expressingEAAC1 (K_(m)=9 μM; V_(max)=1.02 nmol/min/mg protein; FIG. 4A). Similarresults are observed with other cell lines (COS7 and C6 glioma).

EXAMPLE 9 Cell Surface Levels of GTRAPs and Cytoskeletal Stability

To examine changes in the cell surface levels of EAAT4, a cellmembrane-impermeant biotinylation reagent to label cell surface proteinsselectively. Biotinylation of cell surface proteins was performed asdescribed in Duan et al. (Duan, et al., J. Neurosci. 19, 10193-10200(1999)). SOD1 was used to control for total protein and to determinewhether the biotinylation reagent labels proteins in the intracellularcompartment. Densitometry was performed using the NIH Image program.

The total amount of EAAT4 is increased when GTRAP4-41 and GTRAP4-48 areco-expressed (FIG. 5). In contrast the total amount of SOD1, a controlfor total amount of protein loaded, is unaltered or decreased in theGTRAP4-41 and GTRAP4-48 samples, respectively. The majority of the EAAT4is biotinylated, indicating that it is at the cell surface. However thepercentage of total EAAT4 that is at the cell surface remains the samewhen GTRAP4-41 and GTRAP4-48 are co-expressed. Taken together, theseresults indicate that GTRAP4-41 and GTRAP4-48 stabilize/anchor EAAT4 atthe cell membrane, making it less likely to be internalized andsubsequently degraded, rather than causing an increased trafficking ofEAAT4 to the cell surface.

However it is also possible that there is increased expression of thecell's native gene. To address this question cells were treated 48 hrsafter transfection with cycloheximide, an inhibitor of proteinsynthesis. Quantification by densitometry shows that 12 hrs aftertreatment, the EAAT4 protein in HEK-rEAAT4 cells is reduced to 54±0.6%of the level prior to cycloheximide treatment. In contrast, 81±2% and74±1.7% of the EAAT4 protein remains after 12 hrs when GTRAP4-41 andGTRAP4-48 are coexpressed, respectively. These results provide evidencethat GTRAP4-41 and GTRAP4-48 do stabilize EAAT4 at the membrane.

EXAMPLE 10 GTRAP- Glutamate Transport Protein Interactions

To determine whether the EAAT4/GTRAP4-48 interaction is required tomediate the increase in EAAT4 activity, HEK-rEAAT4 cells weretransfected with GTRAP4-48 constructs lacking the last 155 amino acidswhich were pulled out by EAAT4 in the yeast two-hybrid screen. Thecarboxy-terminally truncated GTRAP4-48 had only a modest effect onstimulating EAAT4 activity, indicating that the protein-proteininteraction is responsible for the majority of the increase in uptakeactivity. HEK-rEAAT4 cells were co-transfected with GTRAP4-48 and amyc-tagged cDNA construct encoding the last 77 amino acids of EAAT4 todisrupt the interaction of GTRAP4-48 with full-length EAAT4.Co-expression of this construct inhibits the GTRAP4-48 mediated effectby approximately 25%, but co-expression of a smaller construct (residues1452 to 1578), which lacked the GTRAP4-48 binding domain, has no effect.Taken together these results indicate that the EAAT4/GTRAP4-48interaction plays an important role in the modulation of EAAT4 uptakeactivity.

These results are summarized in FIG. 6. FIG. 6A shows results ofexperiments in which HEK-rEAAT4 cells were transfected with a GTRAP48construct that lacked the C-terminus (domain that interacts with EAAT4).Disruption of the EAAT4/GTRAP48 interaction significantly reduces theGTRAP48-mediated increase in EAAT4 uptake activity (* p<0.05).Disruption of the protein-protein interaction by overexpression of theEAAT4 C-terminus in HEK-rEAAT4 cells transfected with GTRAP48. TheGTRAP48-mediated effect on EAAT4 activity was reduced by ˜25% (**p<0.005; FIG. 6B). Na⁺-dependent L-[³H]-glutamate was assayed intriplicate and values are expressed as the mean±SEM of six independentexperiments. GTRAP41 and GTRAP48 significantly increased glutamateuptake in vivo (* p<0.05; FIG. 6C).

The physiological relevance of GTRAP4-41 and GTRAP4-48 on EAAT4 uptakeactivity in vivo was subsequently examined by the intra-cisternalinjection of HSV amplicon vectors expressing GTRAP4-41 and GTRAP4-48.Cerebellar glutamate uptake was measured 48 hrs after injection andfound to be elevated when GTRAP4-41 and GTRAP4-48 are expressed but notwhen the control HSVlac amplicon vector was injected (FIG. 6C).Dihydrokainic acid (DHK), an inhibitor of GLT-1 mediated glutamatetransport, has no effect on cerebellar glutamate uptake, ruling out anyinvolvement of GLT-1. Unfortunately there is no method to distinguishfunctionally between GLAST, EAAC1 and EAAT4. However it has been haveshown that GTRAP4-41 and GTRAP4-48 do not interact directly with anyother transporter, it is likely that the observed increase in uptake isattributed to an increase in EAAT4 activity. Western blot analysisconfirms increased expression of GTRAP4-41 and GTRAP4-48 in thecerebellum following the injections.

EXAMPLE 11 Clustering of Glutamate Transporter Proteins at Synapses

To examine whether GTRAPs are involved in, or associated with, theclustering of EAAT4 at synapses primary cultures of rat Purkinje cellneurons were examined immunocytochemically. Rat brain sections werestained, as previously described (Furuta et al., Neurosciences 81:1031-1042 (1997)) using the following antibodies: carboxy-terminalanti-EAAT4 (1.5 μg/ml), anti-GTRAP4-41 (127 ng/ml) or anti-GTRAP4-48(132 ng/ml) antibodies. Texas-red and FITC-conjugated secondaryantibodies were used at dilutions of 1:200.

EAAT4 and GTRAP4-41 immunoreactivity is observed throughout the soma anddendrites but is also found to colocalize in distinct clusters. Labelingwith synaptophysin, a presynaptic protein, reveals that 71% of synapsespossessed clusters of EAAT4 and GTRAP4-41 [n=12]. This perisynapticdistribution of GTRAP4-41 correlates with earlier EM studies that showedthat EAAT4 is a perisynaptic protein. Similar studies could not becarried out for GTRAP4-48 due to low level of expression at this earlydevelopmental stage.

EXAMPLE 12

Interaction with Rho: Since GTRAP4-48 possesses areas of homology top115 and PDZRhoGEF, two novel RhoGEFs that selectively activate Rho(Hart et al., J. Biol. Chem. 271, 25452-25458 (1996), Fukuhara et al.,J. Biol. Chem. 274, 5868-5879 (1999)), interaction of GTRAP4-48 with theRho family of GTPases was investigated.

Guanine nucleotide exchange assay. Small G proteins GST-RhoA, GST-CDC42and GST-Rac were expressed in bacterial cells and affinity purified to˜80% purity using a glutathione column. Twenty pmoles of each proteinwere incubated with 100 pmoles GTPγS for 10 min at 30° C. with varyingconcentrations of full-length GTRAP4-48 or p115. The binding reactionswere filtered through BA-85 nitrocellulose and the amount of GTPγS boundto small G protein was quantitated by scintillation counting of thedried filters. The amount of GTPγS that bound to GST-RhoA, GST-Cdc42 andGST-Rac in the presence of full-length GTRAP4-48 or p115 was measured.

GTRAP4-48, like p115, demonstrates a specific guanine nucleotideexchange activity for Rho (FIG. 7). Co-immunoprecipitation assays alsoshow that GTRAP4-48 interacts with the active form (in the presence ofaluminium fluoride) of the Gα₁₃ subunit and therefore, may act as a linkbetween G-protein coupled receptors and their downstream targets.However, unlike p115, regulation of the GTRAP4-48 RhoGEF activity byGα₁₃ nor the stimulation of the GTPase activity of Gα₁₃ by GTRAP4-48could be demonstrated. Rho is known to regulate the remodeling of theactin cytoskeleton through various actin-binding proteins, although themechanism is not yet well characterized (Hall, Science 279, 509-514(1998)).

Since GTRAP4-48 can activate Rho, expression of GTRAP4-48 was studied todetermine if it could induce the reorganization of the actincytoskeleton and whether it alters the distribution of GTRAP4-41, apossible actin binding protein. When GTRAP4-41 is expressed alone thereis a close relationship between actin and GTRAP4-41 at the cell membranebut there are very few organized actin filaments. Conversely, whenGTRAP4-41 and GTRAP4-48 are co-expressed, GTRAP4-41 is found toco-localize with actin in structures that resembled actin-stress fibers,a typical Rho-dependent effect. Overexpression of GTRAP4-48 also inducesthe formation of membrane ruffling and filopodia, suggesting some degreeof cross-talk between the small GTPases, as these are typical Rac andCdc42 dependent effects. These results indicate that there is a closerelationship between GTRAP4-48 and the reorganization of GTRAP4-41 andthe actin cytoskeleton.

EXAMPLE 13 Antisense Treatment with GTRAP3-18

To demonstrate tonic modulation of EAAC1 activity by GTRAP3-18,antisense oligomers were used to lower GTRAP3-18 expression in HEK293cells. Western blot analyses and glutamate uptake assays revealendogenous expression of EAAC1 and GTRAP3-18 protein in HEK cells, butno expression of other transporter subtypes, e.g., GLAST, GLT-1, orEAAT4. Antisense oligomers, targeted to the 5′-GTRAP3-18 transcript,were transfected into HEK293 cells.

Antisense oligomers specifically reduced endogenous GTRAP3-18 proteinlevel (FIG. 8A, gray bars); EAAC1 protein level was not affected.Significantly, glutamate transport activity was concomitantly elevatedwith the reduction of GTRAP3-18 protein level (black bars).

To examine modulation of EAAC1 by GTRAP3-18 in vivo, GTRAP3-18 antisenseoligomers were administered intraventricularly. Sequences for the novelphosphodiester oligonucleotides used were: sense GTRAP3-18:5′-GTGAACCTTGCCCGCTC-3′, antisense GTRAP3-18: 5′-GAGCGGGGCAAGGTTCAC-3′Oligonucleotides (5 μg/μL), separately or in combination wereadministered intraventricularly over 3-11 days, by mini-osmotic pumps(Alza Corp., Palo Alto, Calif.) as described previously (Rothstein etal., (1994)).

Eleven days of antisense treatment resulted in a reduction of GTRAP3-18protein level and a significant increase in cortical glutamate uptake,whereas glutamate uptake was not altered in sense oligomer-treatedanimals (FIG. 8B). The effect was due to increased EAAC1 -mediatedtransport because it was not altered by dihydrokainic acid (DHK), aninhibitor of GLT-1-mediated glutamate transport (Robinson et al. (1998)Neurochem. Int. 33:479-491). In kinetic studies of DHK-insensitive,cortical glutamate uptake from antisense-treated animals, the apparentaffinity for glutamate was increased (antisense K_(m)=10 μM,V_(max)=1.08 nmol/min/mg protein) compared to artifical CSF or sensetreated control animals (control K_(m)=19.7 μM; V_(max)=1.08 nmol/min/mgprotein; FIG. 8C). These results suggest that GTRAP3-18 negativelymodulates EAAC1 glutamate transport activity in vivo.

EXAMPLE 14 Regulation of GTRAP3-18 by Retinoic Acid

Human GTRAP3-18 (JWA protein) was originally identified as a retinoicacid responsive gene. Therefore, retinoic acid was tested for itsability to up-regulate GTRAP3-18 expression and consequently inhibitEAAC1-mediated glutamate transport in HEK293 cells. Retinoic acidinduces a large increase in GTRAP3-18 expression, over a non-toxic doserange from 1-10 μM. A significant decrease in glutamate transportactivity paralleled the increase of GTRAP3-18 protein level (FIG. 9).The loss of transport activity is not due to changes in EAAC1 proteinlevel (FIG. 9A) or the cellular membrane localization of EAAC1 proteinby retinoic acid as examined by fluorescent microscopy. To confirm thatloss of transport activity was specifically due to GTRAP3-18 and not byother factors induced by retinoic acid or direct effects on EAAC1, atruncated EAAC1 cDNA, lacking the last 93 amino acids, was constructed.The truncation corresponded to the region used as bait in yeasttwo-hybrid screening, and was not able to interact with GTRAP3-18.Nevertheless, after transient expression in HEK293 cells, the truncatedEAAC1 transported glutamate. Importantly, retinoic acid treatment doesnot alter activity of the truncated EAAC1 protein; even though GTRAP3-18protein expression was markedly elevated (FIG. 10B). Thus, the loss oftransport activity by retinoic acid was the result of GTRAP3-18induction. Interestingly, truncated EAAC1 has increased glutamatetransport activity compared to wild-type. Truncated EAAC1 had a K_(m) of5.4 μM, which was greater than a three-fold increase in affinitycompared to wild-type EAAC1 (Km=17 μM; FIG. 9C). This could reflect lackof natural inhibition of the truncated protein EAAC1 by endogenousGTRAP3-18 - results similar to the effects of GTRAP3-18 antisensetreatment (FIG. 9E).

To test this hypothesis in vivo, retinoic acid was infusedintraventricularly (1-20 μM; 0-20 pmol/μL). After 4 days of treatment,cortical GTRAP3-18 protein expression was increased in a dose dependentmanner, and this was associated with a significant decrease of totalglutamate uptake (FIG. 9D, top panel). This effect is specifically dueto decreased EAAC1-mediated transport because it was not altered by theglutamate transport inhibitor dihydrokainic acid, at a concentrationthat predominantly effects GLT-1 (Robinson et al. (1998)). Kineticanalysis of DHK-insensitive, cortical glutamate transport from animalstreated four days with intraventricular retinoic acid reveals a 4-folddecrease in affinity compared to control transport (FIG. 9E) which isvery similar to that seen in vitro (FIG. 9B). In addition, retinoic acidinhibition of glutamate transport could be reversed in vivo; chronicintraventricular treatment with antisense GTRAP3-18 oligomer (50-100ng/day, for 7-10 days) blocks the retinoic acid (2.5 μM) induction ofGTRAP3-18, and also blocks the inhibition of glutamate transport seenwith retinoic acid treatment (FIG. 9D, top panel). Retinoic acid had noeffect on glutamate transport by cells expressing GLT-1 or EAAT4.

EXAMPLE 15 Glutamate Transporter Associated Protein PCTAIRE-1

The glutamate transporter EAAT4 possesses high affinity Na⁺-dependenttransport activity, as well as a unique ligand-gated Cl⁻ conductance.Largely located in the somatodendritic compartment of the cerebellarPurkinje cell, altered function of EAAT4 may contribute to thepathogenesis of spinocerebellar ataxia and alcoholic cerebellardegeneration. In an effort to delineate possible regulatory mechanismsof EAAT4, we have identified glutamate transporter associated proteins(GTRAPs). Using the amino terminus of rat EAAT4 as bait in a yeasttwo-hybrid screen, an interacting protein was isolated. Subsequentsequence analysis identified the GTRAP as PCTAIRE-1, a serine/threoninekinase related to the cyclin-dependent kinase 2 (cdk2) family. In vitroand in vivo co-immunoprecipitations from rat cerebelli were performed,confirming specificity of interaction; co-localization of EAAT4 andPCTAIRE-1 within the cerebellum was determined using immunofluorescence.In order to investigate regulatory physiology of the PCTAIRE-1I/EAAT4interaction, co-transfection experiments and pharmacologic manipulationwere carried out. PCTAIRE-1, although a member of the cdk2 family, ispresent mainly in terminally differentiated tissues such as brain. Ithas been shown to interact with members of signal transduction cascades(14-3-3 proteins) and components of cellular protein networks such asp11, a target for annexin II. These data suggest a mechanism by whichEAAT4 may be linked to cellular regulatory machinery via the GTRAPPCTAIRE-1.

Methods. Yeast Two-Hybrid Screening: Screening was performed using theAH109 yeast strain harboring the reporter genes ADE/HIS, as well α- andβ-galactosidase. The initial 60 amino acids of EAAT4 were subclonedin-frame into pGBKT7 (GAL4 binding domain, CLONTECH), and used to screena rat brain cDNA library constructed in pACT2 (GAL4 activation domain,CLONTECH). Following cotransformation and verification of true positivecolonies, DNA sequence analysis was performed. Obtained sequences werecompared to known GENBANK submissions, resulting in identification of atrue postive with with >95% homology to the final 201 amino acids of ratPCTAIRE-1.

Creation of expression constructs: Full length rat PCTAIRE-1 wasisolated from a rat brain cDNA library via PCR amplification usingupstream and downstream primers based on the known PCTAIRE sequence.Products were cloned into pCMVmyc tagged eukaryotic expression vector(CLONTECH), and expression verified by western blotting.

Two PCTAIRE-1 proteins are identified. PCTAIRE-1a is encoded byPCTAIRE-1 nucleic acid sequence, nucleotides 251-452 and 584-1872 (SEQID NO:8) and PCTAIRE-1b is encoded by PCTAIRE-1 nucleic acid sequence,nucleotides 487-1872 (SEQ ID NO:22).

Immunoprecipitations. In-vitro coimmunoprecipitaions were performed onstably transfected HEK cells expressing EAAT4. Vector DNA or myc-taggedPCTAIRE vector was then introduced. Following expression, cells weresolubilized with ice-cold IP buffer and centrifuged to remove cellulardebris. 0.5 ml of supernatant was then incubated with or without 1.5 μganti-c-myc antibody (Boehringer-Mannheim). Complexes were then isolatedusing protein-A Sepharose, washed four times with IP buffer with andwithout Triton X-100, and visualized using SDS-PAGE. EAAT4 westernblotting was performed using affinity purified rabbit polyclonal Ab at1:200 dilution.

In-vitro coimmunoprecipitation of EAAT4 with myc tagged antibody inEAAT4 expressing HEK cells transfected with myc-labeled PCTAIREs isdemonstrated.

In-vivo coimmunoprecipitation was performed using the cerebellum of a 5day-old Sprague-Dawley rat. Homogenization was performed on ice using abuffer containing 20 MM Tris-HCl (pH 7.5), 10% sucrose, 1 mM EDTA, andprotease inhibitors. The homogenate was mixed 1:1 with buffer containing2% Triton X-100, and solubilized for 2 h at 4° C. 0.5 mg of protein wasused for each immunoprecipitation. Antibodies to the carboxy terminalEAAT4 (2.0 μg), as well as antibody to the transporter GLT (2.0 μg) wereused. In addition, blocking peptide was presorbed to EAAT4 Ab to furtherdemonstrate specificity. Western blotting was performed using PCTAIRE-1antibody at 1:200 dilution (Santa Cruz).

In-vivo coimmunoprecipitation of PCTAIRE by EAAT4 is found in neonatalrat cerebellum. A PCTAIRE doublet (62 and 68 kDa) is recovered byimmunoprecipitation with c-terminal EAAT4 Ab, and inhibited bypreabsorption of EAAT4 Ab with blocking peptide.

Transfection of EAAT4 expressing HEK cells with PCTAIRE results indiminished Na⁺-dependent glutamate uptake. HEK cells and EAAT4expressing HEK cells were transfected with 1.0 μg of pCMV PCTAIREplasmid, and allowed to express for 48 hours. Cell monolayers were thenwashed with tissue buffer (50 mM Tris, 320 mM sucrose, pH 7.4). Thecells were then incubated for 4 min at 37° C with 1 mL of eithersodium-(120 mM NaCl, 25 mM NaHCO₃, 5 mM KCl, 2 mM CaCl₂, 1 mM KH₂PO₄, 1mM MgSO₄, 10% glucose and 10 μM glutamate or choline-(120 mM choline-Cl,25 mM Tris-HCl, 5 mM KCl, 2 mM CaCl₂, 1 mM KH₂PO₄, 1 mM MgSO₄, 10%glucose and 10 μM glutamate ) containing buffer. Glutamate uptake assayswere then performed using L-[³H]-Glutamate in the presence and absenceof Na⁺. After rinsing, cells were lysed in 0.1 N NaOH and lysateradioactivity measured using a scintillation counter. Protein contentwas measured and glutamate uptake calculated as the difference betweenNa⁺ containing and sodium free values per mg of protein.

Inhibition of Na⁺-dependent glutamate uptake by PCTAIRE is reversibleusing the cdk2 inhibitor olomucine. HEK cells expressing EAAT4 weretransfected with 1.0 μg of pCMV PCTAIRE plasmid as described above, andallowed to express for 48 hours. Prior to glutamate uptake assay, cellswere treated with 100 μM olomucine for 30 minutes at 37° C. asindicated. Olomocine belongs to a class of cyclin dependent kinaseinhibitors which inhibit activity via competition at the ATP bindingsite.

Immunofluorescence microscopy displays colocalization of EAAT4 andPCTAIRE in the Purkinje cell layer of the rat cerebellum. A five day-oldrat pup was perfusion fixed, the brain extracted, and 25 μm sectionsstained with antibodies to c-terminal EAAT4 (1.5 μg/mL) and PCTAIRE-1(1.5 μ/mL). Prominent double-labeling is evident in the Purkinje celllayer, especially the cell soma, where EAAT4 is known to be presentduring the early postnatal period.

These results indicate that the serine/threonine kinase PCTAIREinteracts with the amino-terminus of the glutamate transporter EAAT4.This interaction results in downregulation of Na⁺-dependent glutamateuptake, and this process is reversible using an inhibitor of cyclindependent kinases. In addition, immunofluorescence reveals that bothEAAT4 and PCTAIRE localize to the cerebellum, particularly the purkinjecell layer. Although PCTAIRE bears homology to the family of cyclindependent kinases involved in proliferation, it is found mainly interminally differentiated tissues such as brain. Other EAAT4 interactingproteins have recently been identified, both of which interact at thecarboxy-terminus, and upregulate glutamate uptake. GTRAP 41 is a newmember of the β-III spectrin family, and is likely an actin-bindingprotein. GTRAP4-48 is a novel RhoGEF that may provide a link between theheterotrimeric G-proteins and small GTP-binding proteins of the Rhofamily. Together with PCTAIRE, these interactors may regulate glutamateuptake through EAAT4.

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. An isolated polynucleotide selected from the group consisting of: (a) a polynucleotide encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:6; (b) a polynucleotide of (a), wherein all T's are replaced by U's; (c) a polynucleotide complementary to the full length polynucleotide of (a); and (d) a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO:5.
 2. An expression vector comprising a polynucleotide of claim
 1. 3. The expression vector of claim 2, wherein the vector is virus-derived.
 4. The expression vector of claim 2, wherein the vector is plasmid-derived.
 5. An isolated host cell comprising a vector of claim
 2. 6. A method for producing a polypeptide comprising the steps of: (a) culturing a host cell of claim 5 under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
 7. The isolated polynucleotide of claim 1, wherein the polynucleotide encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:6.
 8. The isolated polynucleotide of claim 1, wherein the polynucleotide has a nucleotide sequence as set forth in SEQ ID NO:5. 